Retroviral vector particles expressing complement inhibitor activity

ABSTRACT

Modified retroviral vector particles and modified retroviral producer cells producing such particles are provided for facilitating gene therapy procedures involving the transduction of target cells with retroviral vector particles in the presence of complement containing body fluids. The modifications involve genetic alterations to effect the expression by these cells and particles of complement inhibitor activity. The genetic alterations involve the introduction of nucleic acid expression constructs directing the expression of retroviral SU(gp70)/complement inhibitor chimeric proteins into cells from which the producer cells are derived.

FIELD OF THE INVENTION

The present invention relates to gene therapy mediated by thetransduction of primate cells by retroviral vector particles (RVVPs)and, in particular, to the engineering of cells producing RVVPs toprovide RVVPs expressing complement inhibitor activity. Such engineeredparticles are suitable for medical use for the transduction of human andother primate cells without removing the cells from contact with theextracellular fluids of the host organism.

BACKGROUND OF THE INVENTION I. Retroviruses

The Retroviridae virus family encompasses all viruses containing an RNAgenome and producing an RNA-dependent DNA polymerase (reversetranscriptase). The family is divided into three subfamilies: (1)Oncovirinae, including all the oncogenic retroviruses (referred to inthe older literature as "oncornaviruses") and several closely relatednononcogenic viruses (collectively referred to herein as"oncoretroviruses"); (2) Lentivirinae, the "slow retroviruses," such asthe human immunodeficiency virus (HIV) and visna virus; and (3)Spumavirinae, the "foamy" retroviruses that induce persistentinfections, generally without causing any clinical disease. Theretroviruses may also be classified based upon virion morphology as typeB, type C, or type D retroviruses (Weiss et al., (eds), RNA TumorViruses, Cold Spring Harbor Laboratory, NY, 1985, Vol. 1, pp.31-34,46-51, and Vol. 2, pp. 2-7). More detailed descriptions of theretroviruses may be found in Weiss et al., 1985, Volumes 1 and 2;Doolittle, et al., Quart. Rev. Biol., 64:1-29, 1979; and Varmus andBrown, Retroviruses, p. 53-108, in Berg and Howe, (eds), 1989, MobileDNA, American Society for Microbiology, Washington, DC.

In broadest overview, the life cycle of a retrovirus comprises entry ofan infectious retroviral particle into a host cell, integration of thevirus' genetic information into the host cell's genome, and productionof new infectious retroviral particles by the biosynthetic machinery ofthe infected host cell. More specifically, upon entering a cell, aretroviral particle initiates a series of interactive biochemical stepsthat result in the production of a DNA copy of the virus' RNA genome andits integration into the nuclear DNA of the cell. This integrated DNAcopy is referred to as a provirus and can be inherited by any daughtercells of the infected cell like any other gene. Genes contained withinthe integrated provirus may be expressed in the host cell.

All retroviral particles share common morphological, biochemical, andphysical properties, including:

(1) A linear, positive-sense, single-stranded RNA genome composed of twoidentical subunits and making up about 1% of the mass of the virus.

(2) At least three types of proteins encoded by the viral genome, i.e.,gag proteins (the group antigen internal structural proteins), polproteins (the RNA-dependent DNA polymerase and integrase proteins), andenv proteins (the viral envelope protein or proteins). These proteinstogether make up about 60%-70% of the mass of the virus.

(3) Lipid derived from the cell membrane of an infected cell making upabout 30%-40% of the mass of the virus.

(4) Carbohydrate associated with the env proteins, making up about 2-4%of the mass of the virus.

(5) An overall spherical morphology with variable surface projections.

(6) An isocahedral capsid structure containing a ribonucleoproteincomplex within an internal nucleoid or nucleocapsid shell.

In addition to genes encoding the gag, pol, and env proteins, the genomeof the retrovirus includes two long terminal repeat (LTR) sequences, oneat each end of the linear genome. These 5' and 3' LTRs serve to promotetranscription and polyadenylation of viral mRNAs. Adjacent to the 5' LTRare sequences necessary for reverse transcription of the viral genome(the tRNA primer binding site) and for efficient encapsulation of viralRNA into particles (the Psi site). Other genes may also be found betweenthe 5' and 3' LTRs of the retroviral genome.

If heterologous genes are inserted in between the 5' and 3' LTRs of aretroviral genome, which is then packaged into a functional retroviralparticle, the resulting recombinant retroviral particle is capable ofcarrying the heterologous genes into a host cell. Upon integration ofthe recombinant retroviral genome into the host cell's genome as part ofthe proviral DNA, the heterologous genes may be expressed.

These properties and capabilities have led to the development ofretroviral vectors, retroviral packaging and producer cells, andretroviral vector particles (collectively referred to as retroviraltransduction systems) as efficient means of stably introducing exogenousgenes of interest into mammalian cells. Certain retroviruses have beenengineered to produce non-infectious retroviral transduction systemsthat are especially useful in the field of gene therapy. See Anderson,1992; Miller, 1992; Mulligan, 1983; Mann, 1983; Cone and Mulligan, 1984.

II. Gene Transfer by Oncoretroviral Transduction

Retroviral transduction systems of the type discussed above are able tointroduce recombinant nucleic acid molecules into mammalian targetcells, and to efficiently integrate DNA molecules containing some or allof the genetic information (sequence) of the introduced recombinantnucleic acid molecule into the genome of the target cell so that theintroduced genetic material is replicated and is stably and functionallymaintained (and any encoded gene products are expressed) in the cellwithout the danger of the production of replicating infectious virus.See, for example, Ausubel, et al., 1992.

Oncoretroviral vector particles are particularly useful for geneticallymodifying mammalian cells, including human cells, because the efficiencywith which they can transduce target cells and integrate their geneticinformation into the target cell genome is higher than that achievableusing other systems of introducing exogenous genetic material intocells. Other advantages associated with the use of oncoretroviral vectorparticles as gene therapy agents include stable expression oftransferred genes, capacity to transfer large genes, and lack ofcellular cytotoxicity. Additionally, oncoretroviral vector particles maybe constructed so as to be capable of transducing mammalian cells from awide variety of species and tissues.

Successful gene transfer by transduction with a retroviral vectorparticle (RVVP) requires: 1) incorporation of a gene of interest into aretroviral vector; 2) packaging of vector-derived RNA into a RVVP; 3)binding of the RVVP to the target cell, generally via a cell surfacereceptor on the target cell; 4) penetration of at least the RNAmolecules comprising the viral genome into the target cell (generallyassociated with penetration of the RVVP and uncoating of the RVVP); 5)reverse transcription of the viral RNA into pre-proviral cDNA; 6)incorporation of the pre-proviral cDNA into preintegration complexes, 7)translocation of the preintegration complexes into the target cellnucleus, 8) generation of stable proviral DNA by integration of thepre-proviral cDNA into the host genome (typically mediated by the viralintegrase protein); and 9) expression of the gene of interest. In the invivo setting (and in some ex vivo settings), the RVVP must survive inthe extracellular fluids of the host organism in an active state for aperiod sufficient to allow binding and penetration of the host targetcell by the RVVP.

Oncoretroviral Envelope Proteins: The binding of an RVVP to a receptoron a target cell referred to above is mediated by retroviral envelopeproteins. In oncoretroviral virions, as exemplified by the Moloneymurine leukemia virus, the envelope proteins responsible for thisbinding are found as heterodimers of two polypeptides. Thesepolypeptides are both encoded by the env gene and are proteolyticallycleaved by cellular proteases from a common precursor, gp80. The p15Eenvelope protein is a transmembrane polypeptide also referred to in theart as TM. The gp70 envelope glycoprotein (also referred to in the artas the surface or SU protein) associates with p15E through anon-covalent interaction. On a mature retroviral virion, the TM proteinis anchored in the viral membrane and the SU protein is attached to theTM protein and is exposed on the surface of the virion. Large numbers ofTM/SU heterodimers are found on oncoretroviral virions. In general, anoncoretroviral SU protein includes the following regions: (i) asecretory signal or "leader" sequence; (ii) a receptor binding domain;(iii) a hinge or neck region; and (iv) a body portion. The receptorbinding domain of the gp70 protein is essential for the binding of anoncoretroviral virion to a cell surface receptor on a target cell.

Numerous env genes encoding oncoretroviral SU proteins are well known inthe art. Such SU-encoding env genes include those from ecotropic murineleukemia viruses, xenotropic murine leukemia viruses, amphotropic murineleukemia viruses, polytropic murine leukemia viruses, andoncoretroviruses infecting a non-murine species such as feline leukemiaviruses or a gibbon ape leukemia viruses.

Oncoretroviral env genes and the proteins they encode share a highlyconserved structural/functional domains. The nucleotide and, inparticular, the amino acid homologies associated with the conserveddomains of SU proteins allow those of skill in the art to determine thelocations of the various structural/functional domains, even inpreviously uncharacterized SU proteins, by comparison with the sequencesof well characterized SU proteins in which the variousstructural/functional domains are known. Examples of such wellcharacterized oncoretroviral SU sequences include those of the Moloneymurine leukemia virus ecotropic (eco) and xenotropic (xeno) gp70proteins. Eco gp70 has 469 amino acids. Amino acid residues 1-33constitute the leader sequence; amino acid residues 34-263 constitutethe receptor binding domain; amino acid residues 264-312 constitute thehinge region; and amino acid residues 313-469 constitute the bodyportion. Xeno gp70 has 443 amino acids. Amino acid residues 1-30constitute the leader sequence; amino acid residues 31-232 constitutethe receptor binding domain; amino acid residues 233-286 constitute thehinge (or neck) region; and amino acid residues 287-443 constitute thebody portion.

Gene Therapy: There is active research, including clinical trialresearch, on treatment of disease by introduction of genetic materialinto some of the cells of a patient. A variety of diseases may betreated by therapeutic approaches that involve stably introducing a geneinto a cell such that the gene may be transcribed and the gene productmay be produced in the cell. Diseases amenable to treatment by thisapproach include inherited diseases, particularly those diseases thatare caused by a single gene defect. Many other types of diseases,including acquired diseases, may also be amenable to gene therapy.Examples of such acquired diseases include many forms of cancer, lungdisease, liver disease, and blood cell disorders. See Anderson, 1992;Miller, 1992; and Mulligan, 1993.

Delivery of the gene or genetic material into the cell is the firstcritical step in gene therapy treatment of disease. A variety of methodshave been used experimentally to deliver genetic material into cells.Most research has focused on the use of retroviral and adenoviralvectors for gene delivery. As discussed above, RVVPs are particularlyattractive because they have the ability to stably integrate transferredgene sequences into the chromosomal DNA of the target cell and are veryefficient in stably transducing a high percentage of target cells.Accordingly most clinical protocols for gene therapy use retroviralvectors (see, for example, Miller, 1992; and Anderson, 1992).

Most gene therapy protocols involve treating target cells from thepatient ex vivo and then reintroducing the cells into the patient.Patients suffering from several inherited diseases that are each causedby a single gene defect have already received gene therapy treatments.Such treatments generally involve the transduction of the patient'scells in vitro using RVVPs designed to direct the expression oftherapeutic molecules, followed by reintroduction of the transducedcells into the patient. In many cases such treatments have providedbeneficial therapeutic effects.

For many diseases, however, it will be necessary to introduce the geneinto the target cell in situ, because the target cells cannot be removedfrom and returned to the body. In other cases, cells that are removedfrom the patient must be maintained in the presence of body fluids untilbeing returned to the body. Stem cells, particularly hematopoietic stemcells, are an especially important type of target cell for gene therapyof inheritable and acquired blood disorders. Such cells areintrinsically unstable in vitro, and tend to differentiate into cellsthat are less attractive targets for gene therapy, especially when theyhave been washed free of the fluids that surround them in vivo andtransferred into body-fluid-free liquids comprising tissue culture mediaor the like.

Accordingly, it is desirable to transduce stem cells as quickly aspossible, and ex vivo treatment of such cells with RVVPs is best carriedout in the cells natural milieu, i.e., in cells that have not beenwashed or otherwise removed from the body fluids in which they areobtained, e.g., hematopoietic stem cells in bone marrow aspirates. Inthe case of stem cells in bone marrow, current medical procedures forbone marrow transplant involve mixing an ex vivo bone marrow aspirate(which is inevitably obtained as a mixture of bone marrow and blood)with heparin and tissue culture medium. The condition of such cells,that have been removed from the body but kept in diluted or undilutedfluids of their natural milieu, is referred to hereinafter as the "exvivo unwashed state".

III. Complement and Retroviral Vector Particles

A longstanding problem associated with the use of RVVPs as gene therapyvectors in cells in vivo or in cells in the ex vivo unwashed staterelates to the inactivation of many oncoretroviruses (and RVVPs derivedtherefrom) by the body fluids (e.g., blood, bone marrow, lymph) of manyprimates, including Old World monkeys, apes, and humans. Indeed, it hasbeen known for almost two decades that certain oncoretroviruses arerapidly inactivated in human serum (Welsh et al., 1975), as well asserum from nonhuman primates (Welsh et al., 1976). This problem hasprecluded the use of such RVVPs for gene therapy in vivo or in the exvivo unwashed state.

The complement system has long been implicated in the serum mediatedinactivation of oncoretroviruses, as serum deficient in C2, C4 or C8does not cause the detectable release of reverse transcriptase fromoncoretroviral virions (Welsh et al., 1975; Cooper, et al., 1976). Theprotection of active oncoretroviral particles from human complement isthus necessary for the use of the RVVPs to mediate gene therapy in humancells in vivo or in the ex vivo unwashed state. Accordingly, to date,gene transfer by retroviral transduction has been, for the most part,limited to cells that were removed from the extracellular fluids of thehost organism (i.e., ex vivo cells that are not in the ex vivo unwashedstate) and thus were not subjected to complement attack. This limitationhas represented a significant shortcoming of this technology.

The need for methods allowing transduction of primate cells in situ, invivo, or in the ex vivo unwashed state has resulted in the developmentof methods designed to prevent the inactivation of retroviruses by humanand other primate sera. Such methods have included the removal of cellsfrom the extracellular fluids of the host organism, as discussed above,as well as the masking of virion structures that can activate complementactivity by administration of isolated C1s and/or C1q, as discussedbelow under the subheading "The Direct C1 Binding Mechanism".

Significantly, with regard to the present invention, no previous methodsfor allowing transduction of primate cells in situ, in vivo, or in theex vivo unwashed state have included the genetic engineering of cells soas to generate retroviral producer cells producing RVVPs that expresscomplement inhibitor activities.

The Direct C1 Binding Mechanism: Oncoretroviruses that are sensitive tohuman serum can activate the human classical complement pathway by amechanism that involves a unique, antibody independent process. Thisprocess is found in many primates and is generally not present in othermammals (see Cooper, et al., 1976). This mechanism is activated whencomplement component C1 binds to retroviral virions directly andtriggers the classical complement pathway, just as the pathway isnormally activated by an antigen-antibody complex (Bartholomew, et al.,1978). The complement cascade then causes the eventual destruction andelimination of the virus.

Complement component C1 is a large complex protein composed of 3subunits designated C1q, C1s, and C1r. C1q is itself composed of 18polypeptide chains of three different types designated A, B, and C. Sixmolecules each of chains A, B, and C compose the C1q subunit. There aretwo molecules each of the C1s subunit and the C1r subunit that associatewith C1q to form the C1 complement component. The C1q subunit containsmultiple identical binding sites for the complement binding regions ofimmunoglobulin molecules which regions are only exposed upon theformation of an antigen antibody complex. In the classical pathway, thebinding of C1q to these regions of antigen-bound antibody moleculescauses a conformational change in the C1 complex resulting in theenzymatic activation of C1 to yield an active serine protease. The C1sand C1q subunits both have a molecular weight of approximately 85 kDa,and each is cleaved to smaller molecular weight forms of approximately57 kDa and 28 kDa during activation of the C1 complex. The 57 kDa formsof C1s and C1q present in the activated C1 complex contain the proteaseactivity.

In the activation of the classical complement pathway by retrovirusesvia the direct binding of C1, the C1q subunit of C1 binds directly to atleast one site on the retroviral virion. In the case of Moloney murineleukemia virus, the pl5E viral protein has been identified as the C1binding receptor. See Bartholomew, et al., 1978. In contrast to theantibody-mediated classical complement pathway, binding by both the C1qsubunit and the C1s subunit of the C1 complex is required for complementactivation by retroviral particles via this mechanism. Furthermore, theC1s subunit and C1q subunit must bind the viral particle when they arepresent in a functional C1 complex in order for complement activation tooccur by this mechanism. See Bartholomew, et al., 1980.

The C1s subunit is also believed to have a specific binding site forretroviral envelope proteins. It has been shown, using inactiveretrovirus, that prebinding with C1s blocks the subsequent activation ofthe complement cascade by the retrovirus in vitro. See Bartholomew, etal., 1980.

Co-pending U.S. patent application Ser. No. 08/098,944 ("the '944application") , filed Jul. 28, 1993 in the name of James M. Mason andentitled "Pre-binding of Retroviral Vector Particles with ComplementComponents to Enable The Performance of Human Gene Therapy In Vivo,"discuses the use of free C1q or free C1s to block the subsequent bindingand/or activation of the C1 complex by active retrovirus particlesincluding RVVPs.

As described therein, C1s or C1q or a combination thereof are incubatedwith the RVVPs in vitro to form complexes with the particles. Thiscomplex formation blocks the binding sites for C1s and/or C1q andthereby protects the particles from subsequent inactivation or lysiswhen the RVVPs are exposed to complement. As further described therein,blockade of intact C1 binding to RVVPs can be achieved by the use offragments of antibodies that bind the viral envelope proteins of RVVPsbut lack complement binding regions.

As disclosed in the '944 application, the use of these methods improvesthe survival of RVVPs in human serum, but does not completely inhibitretroviral inactivation. The reasons for the incomplete nature of theinhibition of retroviral inactivation by these methods have heretoforebeen unknown, but are now believed to be due in part to the presence ofadditional RVVP-associated factors, other than the C1 binding factorsdiscussed above, that can bind to antibodies in Old World primate andhuman blood and thereby activate the complement system. In accordancewith copending U.S. patent application Ser. No. 08/278,639, entitled"Retroviral Transduction of Cells in the Presence of Complement", whichis being filed concurrently herewith in the names of Russell P. Rother,Scott A. Rollins, William L. Fodor, and Stephen P. Squinto, thesefactors are present in the form of alpha galactosyl epitopes found aspart of retroviral membrane glycoproteins.

The present invention provides new methods and compositions that can beused in conjunction with, or as an alternative to, the blockade ofintact C1 binding to RVVPs. The methods of the present invention thusallow the practice of more efficient gene therapy procedures in vivo andin the ex vivo unwashed state.

Although the C1q and C1s polypeptides and antibody fragments used in thepractice of the invention of the '944 application act to inhibit theactivation of the complement cascade, these polypeptides are notconsidered to be complement inhibitor molecules, as they do not interactwith complement components, but rather act by masking a target viralprotein, thereby preventing the activation of complement component C1 bythe viral protein. This masking of the target protein occurs without anydirect or indirect interaction with any native complement component, andwithout the alteration of the activity of a native complement componentthat is associated with the actions of complement inhibitor molecules.

Along these same lines, in copending U.S. patent application Ser. No.08/278,639, entitled "Retroviral Transduction of Cells in the Presenceof Complement", which is being filed concurrently herewith in the namesof Russell P. Rother, Scott A. Rollins, William L. Fodor, and Stephen P.Squinto, discusses the use of certain galactosyl sugars to block thebinding of certain preformed natural antibodies to RVVPs. Such blockadeof antibody binding can block the activation of the complement system byantigen-bound antibodies, and can thus reduce the inactivation of RVVPsby complement. If desired, these methods of using such sugars as well asother methods discussed in the above-referenced application can be usedin conjunction with the methods of the present invention to effect evengreater reductions of the inactivation of RVVPs by complement. Similarlyto the case of the use of C1q and/or C1s discussed above, the blockadeof antibody binding upon administration of the galactosyl sugars occurswithout any direct or indirect interactions with any native complementcomponents, and without alterations of the activities of any nativecomplement components, which interactions and alterations are associatedwith the actions of complement inhibitor molecules.

Thus, as used herein, the term "complement inhibitor molecule" refers toany molecule that inhibits complement activity by interacting, eitherdirectly or indirectly, with a native complement component or cofactorso as to reduce complement activity, but not to molecules that interferewith complement activation only by masking structures of non-complementcomponents (such as pathogen structures, e.g., retroviral envelopeproteins, or antigen-bound antibodies), and not to molecules thatinterfere with antibody-antigen binding events that can serve toinitiate complement activation. As discussed below, numerous moleculesare known in the art that act to reduce complement activity, forexample, by binding directly to complement components, or bysequestering cofactors such as divalent cations necessary for complementcomponent activity. In accordance with the foregoing discussion, suchmolecules are referred to herein as complement inhibitor molecules.

IV. The Complement System

The complement system acts in conjunction with other immunologicalsystems of the body to defend against intrusion of cellular and viralpathogens. There are at least 25 complement proteins, which are found asa complex collection of plasma proteins and membrane cofactors. Theplasma proteins (which are also found in most other body fluids, such aslymph, bone marrow, and cerebrospinal fluid) make up about 10% of theglobulins in vertebrate serum. Complement components achieve theirimmune defensive functions by interacting in a series of intricate butprecise enzymatic cleavage and membrane binding events. The resultingcomplement cascade leads to the production of products with opsonic,immunoregulatory, and lyric functions.

The complement cascade progresses via the classical pathway or thealternative pathway. These pathways share many components, and, whilethey differ in their early steps, both converge and share the sameterminal complement components responsible for the destruction of targetcells and viruses.

The classical complement pathway is typically initiated by antibodyrecognition of and binding to an antigenic site on a target cell. Thissurface bound antibody subsequently reacts with the first component ofcomplement, C1, as discussed above, includes subunits C1s, C1r, and C1q.

The C1q subunit of C1 mediates the binding of C1 both toantigen-antibody complexes and to retroviruses, although, in the case ofdirect binding to retroviruses, the C1s subunit also has a bindingfunction. The bound C1 undergoes a set of autocatalytic reactions thatresult in the activation of the C1r subunits, which in turnproteolytically activate the C1s subunits, altering the conformation ofC1 so that the active C1s subunits are exposed on the exterior of C1,where they can interact proteolytically with complement components C2and C4.

C1s cleaves C2 and C4 into C2a, C2b, C4a, and C4b. The function of C2bis poorly understood. C2a and C4b combine to form the C4b,2a complex,which is an active protease known as the C3 convertase. C4b,2a acts tocleave C3 into C3a and C3b. C3a is a relatively weak anaphylatoxin. C4ais a stronger anaphylatoxin, and can induce degranulation of mast cells,resulting in the release of histamine and other mediators ofinflammation. C3b has multiple functions. As opsonin, it binds tobacteria, viruses and other cells and particles and tags them forremoval from the circulation. C3b can also form a complex with C4b,C2ato produce C4b,2a,3b, or C5 convertase, which cleaves C5 into C5a(another anaphylatoxin), and C5b. C5b combines with C6 yielding C5b,6,and this complex combines with C7 to form the ternary complex C5b,6,7.The C5b,6,7 complex binds C8 at the surface of a cell membrane. Uponbinding of C9, the complete membrane attack complex (MAC) is formed(C5b-9) which mediates the lysis of foreign cells, microorganisms, andviruses.

A more complete discussion of the classical complement pathway, as wellas a detailed description of the alternative pathway of complementactivation, which pathway has also been implicated in the inactivationof RVVPs by human complement, can be found in Roitt, et al., 1988.

V. Inhibitors of the Complement System

Under normal conditions, homeostatic regulation of the actions ofcomplement proteins is mediated by specific endogenous complementinhibitor molecules (CIMs), that can be found on the surfaces of mosthuman cells. A number of endogenous CIMs have been identified that serveto protect cells from damage mediated by complement. Pathogenicorganisms and viruses are also known to produce CIMs (referred tohereinafter as "microbial CIMs") that are believed to allow thepathogens to evade destruction by the complement of their hostorganisms, and thus act to increase their virulence. Examples of thesevarious types of complement inhibitor molecules are discussed below.

Endogenous CIMs, C3 Inhibitor Proteins--A family of cell-surfaceproteins with shared structural features has been described each ofwhose actions impact on C3b and are referred to as C3 inhibitorproteins.

Decay accelerating factor (DAF or CD55) exists on all cells, includingred blood cells. DAF is a single chain, 70 kDa glycoprotein that islinked to the cell membrane via a glycophosphatidyinositol (GPI) moietywhich inserts into the outer leaflet of the plasma membrane bilayer.

DAF regulates complement activation at the C3 convertase stage bypreventing the assembly of the C3 convertases of both the classical andalternative pathways (Medof et al., 1984; Fujita et al., 1987). Thus,DAF prevents the formation of the anaphylactic cleavage fragments C3aand C5a, in addition to inhibiting amplification of the complementcascade on host cell membranes.

DAF has been shown to act exclusively in an intrinsic manner on cells,protecting only the cell on whose surface it resides while having noeffect on neighboring cells. After extraction from human red bloodcells, DAF reincorporates into cell membranes and is biologicallyactive. Both membrane and secreted forms of DAF have been identified andtheir cDNAs have been cloned and characterized (Moran et al., 1992).

The nucleotide and amino acid sequences for human DAF are set forth inthe Sequence Listings as SEQ ID NO:10.

Membrane cofactor protein (MCP or CD46) exists on all cells except redblood cells. MCP is a type I transmembrane glycoprotein that binds toC3b. MCP acts as a cofactor in the factor I-mediated cleavage of C3b andC4b deposited on self tissue. Therefore, the presence of bound MCPactivates molecules that cleave C3b into inactive fragments, preventingthe potentially cytolytic accumulation of C3b. Nucleotide and amino acidsequences for MCP can be found in Lublin, et al., 1988.

Complement receptor 1 (CR1 or CD35) is found on erythrocytes as well asa select group of leukocytes, including lymphocytes, neutrophils, andeosinophils. CR1 is a 190-280 kDa transmembrane protein that triggersthe proteolytic degradation of membrane bound C3b molecules with whichit comes in contact. It also promotes the clearance of immune complexes.Nucleotide and amino acid sequences for CR1 can be found in Wong, etal., 1985.

Factor H and C4b-binding protein each inhibit the activity ofalternative pathway C3 convertase. Nucleotide and amino acid sequencesfor factor H can be found in Ripoche, et al., 1988; nucleotide and aminoacid sequences for C4b-binding protein can be found in Chung, et al.,1985.

The genes encoding all of these endogenous C3 inhibitory proteins havebeen mapped to the long arm of chromosome 1, band 1q32, and constitute alocus designated the RCA (Regulators of Complement Activity) genecluster. Notable in the molecular structure of these C3 inhibitoryproteins is a common structural motif of approximately 60 amino acidsdesignated the SCR (short consensus repeat), which is normally presentin multiple copies that are not necessarily identical. See Perkins etal. 1988; Coyne, et al., 1992.

The SCR motif of these C3 inhibitory proteins has four conservedcysteine residues and conserved tryptophan, glycine, andphenylalanine/tyrosine residues. The SCRs are usually followed by a longserine/threonine rich region.

In DAF and MCP, the SCRs are known to encode functional domainsnecessary for full complement inhibitory activity (Adams, et al., 1991).DAF is composed of 4 SCRs juxtaposed to a serine/threonine rich regionon the carboxyl terminal side of the SCRs. Most, if not all, of thefunctional domains are reported to reside in SCRs 2 through 4 (Coyne etal., 1992). In SEQ ID NO:10, the 4 SCRs of DAF comprise amino acid 1through amino acid 61 (SCR 1), amino acid 62 through amino acid 125 (SCR2), amino acid 126 through amino acid 187 (SCR 3), and amino acid 188through amino acid 250 (SCR 4), Lublin, et al., 1989.

In addition to these endogenous C3 inhibitor proteins, pathogen C3inhibitor proteins are also known in the art. Examples of these arediscussed below under the subheading "CIMs of Pathogenic Organisms"Genetically modified C3 inhibitor proteins are also known in the art.See, for example, copending U.S. patent application Ser. No. 08/205,508,entitled "Chimeric Complement Inhibitor Molecules, filed on Mar. 3,1994.

Endogenous CIMs, C5b-9 Inhibitor Proteins--The archetypical C5b-9inhibitor protein is the human glycoprotein CD59 (also known as "MACIF,""protectin," or "p18"). The nucleotide and amino acid sequences forhuman CD59 are set forth in the Sequence Listings as SEQ ID NO:11.

CD59 is found associated with the membranes of cells including humanerythrocytes, lymphocytes, and vascular endothelial cells. It serves toprevent assembly of functional C5b-9 membrane attack complexes (MACs)and thus protects cells from complement-mediated activation and/orlysis. CD59 has an apparent molecular mass of 18-21 kilodaltons (kD)and, like DAF, is tethered to the outside of the cell membrane by a GPIanchor. See, for example, Sims et al., U.S. Pat. No. 5,135,916.

CD59 appears to function by competing with C9 for binding to C8 in theC5b-8 complex, thereby decreasing the formation of the C5b-9 membraneattack complex. (Rollins et al., 1990.) CD59 thus acts to reduce bothcell activation and cell lysis by terminal complement MACs. Thisactivity of CD59 is for the most part species-restricted; mostefficiently blocking the formation of MACs under conditions where C8 andC9 are derived from homologous (i.e., human) serum. (Venneker et al.,1992.)

cDNAs encoding CD59 have been cloned and the structure of the CD59 genehas been characterized (Davies, et al., 1989; Okada, et al., 1989;Philbrick, et al., 1990; Sawada, et al., 1989; and Tone, et al., 1992).CD59 has been reported to be structurally related to the murine Ly-6antigens (Philbrick, et al., 1990; and Petranka, et al., 1992). Thegenes encoding these antigens, also known as T-cell activating proteins,are members of the Ly-6 multigene family, and include Ly-6A.2, Ly-6B.2,Ly-6C.1, Ly-6C.2, and Ly-6E.1. The gene encoding the murine thymocyte Bcell antigen ThB is also a member of this family (Shevach, et al. 1989;and Gumley, et al., 1992).

A distinguishing feature of the amino acid sequences of the proteins ofthe Ly-6 family is the arrangement of their cysteine residues. Cysteineresidues of many proteins form a structural element referred to in theart as a "cysteine backbone." In those proteins in which they occur,cysteine backbones play essential roles in determining the threedimensional folding, tertiary structure, and ultimate function of theprotein molecule.

The proteins of the Ly-6 multigene family, as well as several otherproteins share a particular cysteine backbone structure referred toherein as the "Ly-6 motif" For example, the human urokinase plasminogenactivator receptor (uPAR; Roldan, et al., 1990) and one of several squidglycoproteins of unknown function (Sgp2; Williams, et al., 1988) containthe Ly-6 motif.

Subsets of proteins having the Ly-6 motif can be identified by thepresence of conserved amino acid residues immediately adjacent to thecysteine residues. Such conservation of specific amino acids within asubset of proteins can be associated with specific aspects of thefolding, tertiary structure, and ultimate function of the proteins.These conserved patterns are most readily perceived by aligning thesequences of the proteins so that the cysteine residues are in register.

As discussed fully in copending U.S. patent application Ser. No.08/105,735, filed August 11, 1993, by William L. Fodor, Scott Rollins,Russell Rother, and Stephen P. Squinto, and entitled "ComplementInhibitor Proteins of Non-human Primates", the relevant portions ofwhich are incorporated herein by reference, and in Rother, et al., 1994,a series of non-human primate C5b-9 inhibitory proteins have beenidentified which are characterized by a cysteine backbone structurewhich defines a specific subset of the general Ly-6 motif.

Specifically, these non-human primate CIPs include polypeptidescomprising a cysteine backbone with a Ly-6 motif characterized by theformula:

    Cys-X.sub.2 -Cys-X.sub.6-9 -Cys-X.sub.5 -Cys-X.sub.6 -Cys-X.sub.12 -Cys-X.sub.5 -Cys-X.sub.17 -Cys-X.sub.0 -Cys-X.sub.4 -Cys. (1)

In addition, the non-human primate C5b-9 inhibitory proteins includeamino acid sequences conforming to the following formula:

    Cys-X.sub.2 -Cys-Pro-X.sub.5-8 -Cys-X.sub.4 -Asn-Cys-X.sub.5 -(Thr or Ser) -Cys-X.sub.11 -(Gln or Arg)-Cys-X.sub.4 -(Asn or Asp)-Cys-X.sub.17 -Cys-X.sub.0 -Cys-X.sub.4 -Cys.                           (2)

In both formulas; the X in X_(n) indicates a peptide containing anycombination of amino acids, the n in X_(n) represents the length inamino acid residues of the peptide, and each X at any position can bethe same as or different from any other X of the same length in anyother position. C5b-9 inhibitor proteins of viral origin are also knownin the art, examples of which are discussed below under the subheading"CIMs of Pathogenic Organisms".

Vitronectin (S-protein) is another terminal complement inhibitorprotein. A serum glycoprotein found in plasma both in native and inpartially proteolyzed form, it inhibits the lytic activity of themembrane attack complex of complement, by binding to nascent C5b-7complexes, rendering them unable to bind to C8 and C9 (Tschopp, et al.,1988).

C1 inhibitor is a plasma glycoprotein that inhibits the activity of theC1 component of the classical pathway of complement via inhibition ofthe proteolytic activities of the C1r and C1s subunits of C1. C1inhibitor protein migrates in SDS gels at an apparent Mr of 104,000, buthas an actual Mr of approximately 76,000. This complement andcoagulation inhibitor acts by forming a peptidyl bond with a targetproteinase resulting in an inactive complex (see Eldering, 1992).

CIMs of Pathogenic Organisms--Pathogens known to produce CIMs includemicrobial pathogens such as Entamoeba histolytica, which causesamebiasis; Trypanosoma cruzi, which causes Chagas' disease; certainstrains of group A streptococci; Salmonella choleraesuis, variousstrains of which can produce a number of disease states, includingenteric fever and typhoid fever; Yersinia enterocolitica, which causesyersiniosis; vaccinia virus; Herpes simplex virus types I and II; andHerpesvirus saimiri, which causes disease in monkeys (Braga, et al.,1992; Norris, et al., 1991; Hong, et al., 1990; Heffernan, et al., 1992;Bliska, et al., 1992; Isaacs, et al., 1992; McNearney; et al., 1987;Albrecht and Fleckenstein, 1992; Cooper, 1991; Gooding, 1992; andRother, et al., 1994). It is likely that other pathogens, includingnon-microbial pathogens such as worms, produce as yet unidentified CIMs.In addition to those described in general terms above, examples ofspecific microbial CIMs are reviewed below.

Herpesvirus saimiri (HVS), a T-lymphotrophic tumor virus of New Worldprimates, expresses various complement inhibitor molecules. CCPH isfound as both a membrane glycoprotein (mCCPH) and a secreted derivative(sCCPH).

As discussed fully in copending PCT application Serial No. PCT/US93/00672, filed Jan. 12, 1993, by Bernhard Fleckenstein andJens-Christian Albrecht, and entitled "Complement Regulatory Proteins ofHerpesvirus Saimiri", the relevant portions of which are incorporatedherein by reference; and in Rother, et al., 1994; a protein of theherpesvirus saimiri having C5b-9 inhibitory activity has been discovered(referred to herein as "HVS-15"). This viral protein has the Ly-6 motifwhich is characteristic of the non-human primate C5b-9 inhibitoryproteins discussed above, i.e., its structure is described by formulas(1) and (2) above.

Herpesvirus Saimiri also expresses a C3 inhibitor protein referred to asCCPH. The structural layout of CCPH is similar to those of CD55 and CD46and comprises SCR domains. CCPH is found as both a membrane glycoprotein(mCCPH) and a secreted derivative (sCCPH). See, for example, Albrecht,et al., 1992. The nucleotide and amino acid sequences for mCCPH andsCCPH are set forth in the Sequence Listings as SEQ ID NO:12 and SEQ IDNO:13, respectively.

The vaccinia virus complement inhibitor protein VCP has been shown toprevent antibody-dependent complement enhanced neutralization ofinfectivity and to contribute to viral virulence (Isaacs, et al., 1992).

Herpes simplex virus glycoproteins gC-1 and gC-2 have been shown toinactivate complement component C3b, and the presence of either of theseglycoproteins in virions has been shown to provide protection againstcomplement-mediated neutralization of viral infectivity (McNearney, etal., 1987).

The M protein of group A streptococci is a factor that has been shown tobe required for bacterial virulence, and has also been shown to inhibitalternative C3 convertase and classical C5 convertase, two importantenzymatic activities needed for the formation of active terminalcomplement components (Hong, et al., 1990).

The TraT protein of Escherichia coli has been shown to be thedeterminant of the serum resistance conferred by the E. coli R factor,which enhances the virulence of this bacterium (Pramoonjago, et al.,1992).

The rck gene of Salmonella choleraesuis serotype typhimurium (also knownas Salmonella typhimurium) has been shown to be the determinant of theserum resistance conferred by the Salmonella virulence plasmid(Heffernan, et al., 1992).

Other CIMs--In addition to these endogenous and microbial CIMs, a numberof other proteins are known that can inhibit the complement system.

Cobra venom factor (CoVF or CVF) is a C3b-like molecule found in cobravenom that is resistant to C3b inactivating factors (Leventhal, et al.,1993). This C3b analog combines with components of the alternativecomplement pathway to form a highly stable enzyme complex which exhibitshigh levels of C3 convertase activity and causes massive consumption ofC3, terminal complement components, and other factors. This activityresults in the depletion of complement components and the consequentexhaustion of the complement cascade. The end result of CVF action in abody fluid is thus to block terminal complement activity until morecomplement components make their way into the complement depleted bodyfluid.

Antibodies reactive with complement components have the potential toblock complement component action and thereby to inhibit complementactivity. Such complement inhibitory blocking antibodies include themonoclonal antibodies against human C5b-9 proteins discussed in U.S.Pat. No. 5,135,916, issued Aug. 4, 1992. In addition to nativeantibodies, antigen binding fragments (e.g., Fab' preparations) of suchimmunoglobulins, as well as recombinantly expressed antigen bindingproteins, including immunoglobulins, chimeric immunoglobulins,"humanized" immunoglobulins, antigen binding fragments of suchimmunoglobulins, single chain antibodies, and other recombinant proteinscontaining antigen binding domains derived from immunoglobulins, all ofwhich can be prepared by methods well known in the art, can be used asCIMs.

Various non-protein CIMs are also known in the art. See copending U.S.patent application Ser. No. 08/278,550, entitled "RetroviralTransduction of Cells Using Soluble Complement Inhibitors", which isbeing filed concurrently herewith in the names of Russell P. Rother,Scott A. Rollins, James M. Mason, and Stephen P. Squinto.

SUMMARY OF THE INVENTION

In view of the foregoing state of the art, it is an object of thepresent invention to facilitate the use of RVVPs to efficientlytransduce the cells of a primate patient, e.g., a human patient, uponadministration of the RVVPs to cells in contact with the body fluids ofthe patient. Since the RVVPs of the invention and those used in thepractice of the invention are generally used to effect gene therapy,such RVVPs preferably contain an exogenous gene operably linked to apromoter effecting the expression of the gene and operably linked to aportion of the vector which recombines with DNA in the genome of thepatient.

It is an additional object of the present invention to providepharmaceutical agents for gene therapy in primates, and to providearticles of manufacture containing such agents.

In order to achieve these an other objects, the invention providesmodified retroviral vector particles and modified retrovital producercells producing such particles. The modifications involved in makingsuch particles and cells comprise genetic alterations to effect theexpression by these cells and particles of complement inhibitoractivity. The genetic alterations comprise the introduction of nucleicacid expression constructs directing the expression of retroviral SU(gp70)/complement inhibitor chimeric proteins into cells from which theproducer cells are derived.

The invention thus provides Retroviral vector particles expressing acomplement inhibitor activity. The complement inhibitor activityexpressed by the retroviral vector particle is a DAF activity, a CCPHactivity, a CD59 activity, or an activity provided by another complementinhibitor molecule.

The invention also provides methods for transducing a cell in thepresence of a body fluid with a retroviral vector particle comprisingadministering the retroviral vector particle of the invention to atarget cell. In a preferred embodiment, the target cell is in contactwith a body fluid. In a further preferred embodiment, the body fluid isa heparin decoagulated bone marrow aspirate diluted in tissue culturemedium. In another preferred embodiment the body fluid is selected fromthe group consisting of blood, plasma, or serum.

The invention also provides pharmaceutical preparations comprising aretroviral vector particle expressing a complement inhibitor activity ina solution suitable for injection, e.g., intravenous injection. Inaccordance with the invention, such pharmaceutical preparations may bedistributed in the form of an article of manufacture comprising thepharmaceutical preparation and a label indicating that thepharmaceutical preparation is to be used to provide gene therapy to apatient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate the protection of RVVPs by envelope/CIM fusionproteins. RVVP inactivation by human serum or human whole blood wasmeasured using conventional LXSN RVVPs (as controls) and LXSN RVVPsprepared in packaging cells expressing examples of the envelope/CIMfusion proteins of the invention.

FIG. 1 shows results obtained using human serum and LXSN RVVPs preparedin the LXSN/DAF, LXSN/CCPH, and LXSN/CD59 clones that produced the mostresistant RVVPs for each chimeric gp70 type tested. Also shown forcomparison are results obtained with a DAF clone, LXSN3/DAF34, producingless resistant RVVPs which were also tested in experiments using wholeblood.

FIG. 2 shows results obtained using human whole blood and LXSN RVVPsprepared in packaging cells of clone LXSN3/DAF34.

The foregoing drawings, which are incorporated in and constitute part ofthe specification, illustrate certain embodiments of the invention, andtogether with the description, serve to explain the principles of theinvention. It is to be understood, of course, that both the drawings andthe description are explanatory only and are not restrictive of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to gene therapy using retroviral vectorparticles.

I. Retroviral Vector Particles and Packaging Cells for Making SuchParticles

General discussions of packaging cells, retroviral vector particles andgene transfer using such particles can be found in various publicationsincluding PCT Patent Publication No. WO 92/07943, EPO Patent PublicationNo. 178,220, U.S. Pat. No. 4,405,712, Gilboa, 1986; Mann, et al., 1983;Cone and Mulligan, 1984; Eglitis, et al., 1988; Miller, et al., 1989;Morgenstern and Land, 1990; Eglitis, 1991; Miller, 1992; Mulligan, 1993,and Ausubel, et al., 1992.

The packaging cells of the invention are produced by 1) constructing oneor more expression vectors comprising recombinant nucleic acid moleculesencoding the retroviral pol, env, and gag proteins (hereinafter referredto as "packaging vectors"); 2) constructing a chimeric vector directingthe expression of a chimeric SU(gp70)/CIM protein; 3) introducing theone or more packaging vectors and the chimeric vector into culturedcells, typically, mammalian cells; and 4) obtaining the desiredpackaging cells by selecting those cells of the culture which stablyexpress proteins encoded by the one or more packaging vectors and thechimeric vector.

The manipulation of retroviral nucleic acids to construct packagingvectors and packaging cells is accomplished using techniques known inthe art. See Ausubel, et al., Volume 1, Section III (units9.10.1-9.14.3), 1992; Sambrook, et al., 1989; Miller, et al., 1989;Eglitis, et al., 1988; U.S. Pat. Nos. 4,650,764, 4,861,719, 4,980,289,5,122,767, and 5,124,263; as well as PCT Patent Publications Nos. WO85/05629, WO 89/07150, WO 90/02797, WO 90/02806, WO 90/13641, WO92/05266, WO 92/07943, WO 92/14829, and WO 93/14188.

These manipulations typically involve the use of DNA copies of the gag,pol, and env retroviral genes cloned in plasmid vectors. Such plasmidvectors containing retroviral genes can be prepared by, for example,isolation of DNA copies of the viral genome from the cytoplasm ofinfected cells (using, for example, the method of Hirt, 1967),restriction digestion of the DNA copies of the viral genome (or PCRamplification of regions of interest of the DNA, generally followed byrestriction digestion of the PCR product) to produce desired fragments,and multiple rounds of subcloning of the fragments, along with fragmentscontaining suitable selectable marker and origin of replicationsequences, to produce operable packaging vectors.

Multiple rounds of subcloning are used because it has been found thatthe typical bacterial cells used as plasmid hosts in subcloning, e.g.,E. coli, tend to create deletions in the nucleotide sequences of newlyinserted retroviral insert fragments when such fragments comprise morethan about 4 kbp. Accordingly, construction of the final packagingvector proceeds more efficiently if small retroviral insert fragments(on the order of less than about 4 kbp) are sequentially assembled inthe plasmid through multiple rounds of subcloning.

To form the packaging cells, the packaging vector or vectors and thechimeric vector are introduced into suitable host cells. Examples ofsuch cells are found in, for example, Miller and Buttimore, Mol. CellBiol., 6:2895-2902, 1986; Markowitz, et al., J. Virol., 62:1120-1124,1988; Cosset, et al., J. Virol., 64:1070-1078, 1990; U.S. Pat. Nos.4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263, and PCTPatent Publications Nos. WO 85/05629, WO 89/07150, WO 90/02797, WO90/02806, WO 90/13641, WO 92/05266, WO 92/07943, WO 92/14829, and WO93/14188. Preferred host cells are from the NIH/3T3 mouse embryofibroblast line, e.g. ATCC CRL 1658 cells, or the mouse embryofibroblast cell line PA317, ATCC CRL 9078.

Once a packaging cell line has been established, the next step is togenerate "producer cells" by introducing retroviral vectors into thepackaging cells. Examples of such retroviral vectors are found in, forexample, Korman, et al., 1987, Proc. Natl. Acad. Sci. USA, 84:2150-2154;Miller and Rosman, Biotechniques, 7:980-990, 1989; Morgenstern and Land,1990; U.S. Pat. Nos. 4,405,712, 4,980,289, and 5,112,767; and PCT PatentPublications Nos. WO 85/05629, WO 90/02797, and WO 92/07943. Theretroviral vector includes a psi site and one or more exogenous nucleicacid sequences selected to perform a desired function, e.g., anexperimental, diagnostic, or therapeutic function. These exogenousnucleic acid sequences are flanked by LTR sequences which function todirect high efficiency integration of the sequences into the genome ofthe ultimate target cell.

II. Gene Transfer for Gene Therapy

The many applications of gene therapy are well known and have beenextensively reviewed (see, for example, Boggs, 1990; Kohn, et al., 1989;Lehn, 1990, Verma, 1990; Weatherall, 1991; and Felgner and Rhodes,1991).

A variety of genes and DNA fragments can be incorporated into RVVPs,including the RVVPs of the invention, for use in gene therapy. These DNAfragments and genes may direct the expression of RNA and/or proteinmolecules that render them useful as therapeutic agents. Proteinencoding genes of use in gene therapy include those encoding varioushormones, growth factors, enzymes, lymphokines, cytokines, receptors,and the like.

Among the genes that can be transferred in accordance with the inventionare those encoding polypeptides that are absent, are produced indiminished quantities, or are produced in mutant form in individualssuffering from a genetic disease. Other genes of interest are those thatencode proteins that, when expressed by a cell, can adapt the cell togrow under conditions where the unmodified cell would be unable tosurvive, or would become infected by a pathogen. Genes that targettransduced cells for destruction are useful for the treatment ofneoplasias. Genes encoding proteins that have been engineered tocircumvent a metabolic defect are also suitable for transfer into thecells of a patient using the methods and compositions of the presentinvention. Such genes include the transmembrane form of CD59 discussedin copending U.S. patent application Ser. No. 08/205,720, filed Mar. 3,1994, entitled "Terminal Complement Inhibitor Fusion Genes and Proteins"and copending U.S. patent application Ser. No. 08/206,189, filed Mar. 3,1994, entitled "Method for the Treatment of Paroxysmal NocturnalHemoglobinuria".

In addition to protein-encoding genes, the present invention can be usedto introduce nucleic acid sequences directing the expression ofmedically useful RNA molecules into cells. Examples of such RNAmolecules include anti-sense molecules and catalytic molecules, such asribozymes.

In order to expedite rapid transduction by eliminating the need to waitfor target cells to divide, and to allow transduction of cells thatdivide slowly or not at all, the use of retroviral transduction systemsproducing RVVPs that can transduce non-dividing cells may be preferred.Such transduction systems are disclosed in copending U.S. patentapplications Ser. Nos. 08/181,335 and 08/182,612, both entitled"Retroviral Vector Particles for Transducing Non-Proliferating Cells"and both filed Jan. 14, 1994.

III. Preparation and Administration of Complement Resistant RVVPs.

The present invention provides new RVVPs for administration to the bodyfluids of a patient. Significantly, the present invention allows for theuse of more practical protocols for RVVP administration. It does so byeliminating the need to remove the target cells from body fluids priorto administration of the RVVPs. Specifically, in accordance with theinvention, engineered RVVPs are administered to cells while those cellsare in contact with body fluids such as blood, plasma, serum, lymph, thefluids making up bone marrow, and the like.

As discussed above, oncoretroviruses have an envelope SU protein thatcontains a receptor binding region. Applicants have found that RVVPsderived from oncoretroviruses can be made resistant to inactivation bycomplement in the body fluids of a patient if the receptor bindingregion of the retrovirus SU protein, which may be amphotropic,ecotropic, polytropic, or xenotropic, among other types, is modifiedsuch that at least a portion of the receptor binding region of theenvelope protein is removed and replaced with a complement inhibitormolecule. Thus, there is provided a retroviral packaging vector(chimeric vector) wherein at least a portion of the polynucleotidesegment which encodes the receptor binding region of the envelopeprotein of the retrovirus has been deleted and replaced with apolynucleotide segment encoding a complement inhibitor protein.

In the preferred embodiments, the retrovirus from which the RVVPs of theinvention are derived is a murine leukemia virus, with the Moloneymurine leukemia virus being most preferred. In accordance with theinvention, such RVVPs can be made resistant to inactivation bycomplement if a portion of the SU protein is deleted and replaced with acomplement inhibitor molecule.

In general, a retroviral SU protein includes the following regions: (i)the secretory signal or "leader" sequence; (ii) the receptor bindingdomain; (iii) the hinge or neck region; and (iv) the body portion.Preferably, at least a portion of the polynucleotide segment encodingthe receptor binding domain of the SU protein is deleted and replacedwith a polynucleotide segment encoding a complement inhibitor molecule.More preferably, a polynucleotide segment encoding the entire receptorbinding domain of the SU protein is deleted and replaced with apolynucleotide segment encoding a complement inhibitor molecule. Inanother embodiment, a polynucleotide segment encoding the entirereceptor binding domain of the SU protein, plus all or a portion of theDNA (RNA) encoding the hinge region of the SU protein is deleted andreplaced with a polynucleotide segment encoding a complement inhibitorprotein.

The SU protein may be derived from an ecotropic murine leukemia virus, axenotropic murine leukemia virus, an amphotropic murine leukemia virus,or a oncoretrovirus infecting a non-murine species such as the gibbonape leukemia virus. The mature ecotropic gp70 (eco gp70) protein has 469amino acids (SEQ ID NO:7). Amino acid residues 1-33 constitute theleader sequence; amino acid residues 34-263 constitute the receptorbinding domain; amino acid residues 264-312 constitute the hinge region;and amino acid residues 313-469 constitute the body portion. Preferably,a polynucleotide segment encoding some or all of amino acid residues 34to 263 (i.e., the receptor binding domain) is removed and replaced witha polynucleotide segment encoding a complement inhibitor protein.

Xenotropic gp70 (xeno gp70) protein has 443 amino acid residues (SEQ IDNO:8). Amino acid residues 1-30 constitute the leader sequence; aminoacid residues 31-232 constitute the receptor binding domain; amino acidresidues 233-286 constitute the hinge (or neck) region; and amino acidresidues 287-443 constitute the body portion. Preferably, apolynucleotide segment encoding some or all of amino acid residues 31 to232 is removed and replaced with a polynucleotide segment encoding acomplement inhibitor protein.

Nucleic acid molecules encoding a variety of CIMs can be used to protectRVVPs from complement attack. Those CIMs include the molecules discussedabove under the heading "Inhibitors of the Complement System". PreferredCIMs are DAF, CCPH, and CD59.

The CIMs can be used to protect a variety of RVVPs. Preferred RVVPs arethose suitable for gene therapy, including the LXSN particles discussedbelow in the examples. The RVVPs will include a gene or other nucleicacid sequence whose transfer is desired.

In accordance with the invention in certain of its aspects, RVVPs areprepared using producer cells which contain plasmids encoding fusionproteins containing viral envelope and CIM sequences. Such RVVPs, whenso prepared, express the activities of at least one CIM and thus areresistant to inactivation by complement. RVVPs that are less sensitiveto inactivation by complement as a result of genetic engineering ofcells from which producer cells are derived are referred to herein ascomplement resistant RVVPs or crRVVPs.

In general, to form packaging cells in accordance with the invention,the plasmid or plasmids encoding the chimeric SU(gp70)/CIM fusionproteins of the invention are introduced into host cells, either beforeor after the introduction of the packaging vector or vectors describedabove. The producer cells of the invention are prepared by theintroduction of a retroviral vector into the packaging cells of theinvention.

The producer cells of the invention are used to produce crRVVPs byculturing of the cells in a suitable growth medium. If desired, theparticles can be harvested from the culture and administered to thetarget cells which are to be transduced, or the producer cells can begrown together with the target cells. The growth of producer cellstogether with target cells can be accomplished by co-culture of thecells in vitro, or, when desired, the producer cells are co-culturedtogether with the target cells by implantation of the producer cells inthe patient.

In particular, gene therapy may be carried out by a procedure in which aretroviral producer cell (i.e., an engineered cell producing crRVVPs) isimplanted into the body of the patient to be treated. This may be aparticularly desirable procedure in the treatment of certain cancers.Recent in vivo studies have demonstrated that procedures involving theimplantation of producer cells into rat solid tumors can effectivelydeliver RVVPs to adjacent cells (Culver et al., 1992). In one variationof such procedures, producer cells are engineered to express the herpessimplex virus thymidine kinase (HSVTK) gene. Treatment of a patient withganciclovir post-implantation will kill the HSVTK expressing producercells as well as any immediately surrounding cells, which, in suchprocedures, will be tumor cells.

In related studies, producer cells injected into the brain of rats ormonkeys were shown to survive for approximately 15 days withoutproliferating (Ram et al., 1993). The survival of xenogeneic producercells in the primate brain is not surprising considering that the brainis an immunoprivileged site relative to complement activity (Widner andBrundin, 1988) and therefore, hyperacute rejection (HAR) commonlyassociated with xenotransplants into primates is less likely to occur inthe brain. HAR of xenografts in primates normally occurs within minutesof transplantation due to the activation of the classical complementpathway by preexisting antibodies to alpha-galactosyl epitopes found onthe surface of mammalian cells excluding man, apes and Old World monkeys(Galili et al., 1987; and Neethling et al., 1994).

Accordingly, when implantation is performed, the producer cells may beof the type described in copending U.S. patent application Ser. No.08/278,282, entitled "Methods for Reducing Hyperacute Rejection ofXenografts", which is being filed concurrently herewith in the names ofMauro S. Sandrin, William L. Fodor, Russell P. Rother, Stephen P.Squinto, and Ian F. C. McKenzie. They also may be of the type describedin copending U.S. patent application Ser. No. 08/278,639, entitled"Retrovital Transduction of Cells in the Presence of Complement", whichis being filed concurrently herewith in the names of Russell P. Rother,Scott A. Rollins, William L. Fodor, and Stephen P. Squinto.

The crRVVPs of the invention are substantially protected frominactivation upon exposure to the body fluids of an organism of thepatient's species, e.g. incubation in whole blood or in 40% serum. An atleast 5% reduction will, in general, comprise a "substantial reduction".Smaller reductions are also considered "substantial" if they represent astatistically significant reduction; i.e., a reduction that, whenobtained in replicate assays and analyzed by a conventional statisticaltest, such as the student's T test, will give a probability value, p,which is less than or equal to 0.05, and, preferably, less than or equalto 0.015.

The substantial nature of such apparently small reductions is due to thehuge numbers of RVVPs that can be prepared using conventional methods.RVVP titers of greater than 10⁹ RVVPs per ml can be prepared byconcentration (e.g., by tangential flow filter concentration) of RVVPcontaining supernatants obtained using retroviral transduction systemsknown in the art.

As an example, a 100% inactivation may be obtained in the presence of abody fluid, and a 1% reduction in RVVP inactivation may be achievedusing a crRVVP, compared to an unprotected RVVP, i.e., an RVVP notengineered to express the activity of a CIM. In such a case, if 1 ml ofa 10⁹ RVVP per ml preparation is administered to the body fluid, noRVVPs will be present when unprotected RVVPs are administered, and tenmillion RVVPs will be present in the body fluid when the RVVPs of theinvention are administered.

The crRVVPs of the invention can be used for ex vivo gene therapy inaccordance with various techniques known in the art. In general terms,these techniques involve the removal of target cells of interest from apatient, incubation of the target cells with the retroviral vectorparticles, and reintroduction of the transduced target cells into thepatient. Various procedures can be applied to the target cells whilethey are in the ex vivo state, including selection of subsets of thetarget cells prior to transduction, isolation of transduced cells,selection of subsets of isolated, transduced cells, propagation oftarget cells either before or after transduction, in cases where thecells are capable of proliferation, and the like.

Delivery of nucleic acid molecules of interest may also be accomplishedex vivo or in vivo by administration of the retroviral vector particlesof the invention to a patient. In particular, in accordance with theinvention, the crRVVPs can be administered to the target cells while thecells are bathed in body fluids. Specifically, the crRVVPs may beadministered to the target cells via administration to the body fluidsbathing cells in the ex vivo unwashed state using otherwise conventionalprotocols for ex vivo transduction of target cells, or may beadministered to body fluids in vivo. In such in vivo applications, theinjection of crRVVPs directly into solid tissues is considered to beadministration to body fluids, as the cells in solid tissues are bathedin interstitial fluids, and the crRVVPs enter the target cells followingmixture with such fluids.

In connection with such in vivo or ex vivo administration, retroviralvector particles can be pre-treated in accordance with the proceduresdiscussed in co-pending application Ser. No. 08/098,944, filed Jul. 28,1993, in the name of James M. Mason and entitled "Pre-binding ofRetroviral Vector Particles with Complement Components to Enable ThePerformance of Human Gene Therapy In Vivo." Also, the proceduresdescribed in copending U.S. patent application Ser. No. 08/278,550,entitled "Retroviral Transduction of Cells Using Soluble ComplementInhibitors", which is being filed concurrently herewith in the names ofRussell P. Rother, Scott A. Rollins, James M. Mason, and Stephen P.Squinto and in copending U.S. patent application Ser. No. 08/278,639,entitled "Retroviral Transduction of Cells in the Presence ofComplement", which is being filed concurrently herewith in the names ofRussell P. Rother, Scott A. Rollins, William L. Fodor, and Stephen P.Squinto can be used in connection with such administration.

The administration of the crRVVPs can be performed locally, e.g., byaerosol, transmucosal, or transdermal delivery, or, more typically, by asystemic route, e.g., orally, intravenously, intraperitoneally,intramuscularly, transdermally, intradermally, subdermally,transmucosally, or intrathecally. For systemic administration, injectionis preferred.

IV. Pharmaceutical Compositions

The crRVVPs of the invention can be formulated as pharmaceuticalcompositions. Such compositions will generally include apharmaceutically effective carrier, such as saline, buffered (e.g.,phosphate buffered) saline, Hank's balanced salts solution, Ringer'ssolution, dextrose/saline, glucose solutions, and the like. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required, such as, tonicity adjusting agents, wettingagents, bactericidal agents, preservatives, stabilizers, and the like.See, for example, Remington's Pharmaceutical Sciences, Mack PublishingCompany, Philadelphia, Pa., 17th ed., 1985.

The pharmaceutical compositions are suitable for use in a variety ofdrug delivery systems. Langer, Science, 249:1527-1533, 1990, reviewsvarious drug delivery methods currently in use. In some cases, the drugdelivery system will be designed to optimize the biodistribution and/orpharmacokinetics of the delivery of the retroviral vector particles.See, for example, Remington's Pharmaceutical Sciences, supra, Chapters37-39. For example, the particles can be incorporated in vesiclescomposed of substances such as proteins, lipids (for example,liposomes), carbohydrates, or synthetic polymers. See, for example,Langer, 1990, supra.

In certain preferred embodiments, the invention also provides articlesof manufacture consisting of pharmaceutical compositions that contain(a) crRVVPs and/or producer cells, and (b) packaging material indicatingthat the pharmaceutical composition is to be used to effect genetherapy.

The pharmaceutical compositions of the invention can be administered ina variety of unit dosage forms. The dose will vary according to, e.g.,the particular crRVVP or producer cell, the manner of administration,the particular disease being treated and its severity, the overallhealth and condition and age of the patient, and the judgment of theprescribing physician. Dosage levels for human subjects are generallybetween about 10⁶ and 10¹⁴ colony forming units of retroviral vectorparticles per patient per treatment. Producer cells are administered insufficient numbers to produce therapeutic levels of crRVVPs, e.g., atleast about 10³ -10⁴ producer cells.

In terms of clinical practice, the compositions and methods of thepresent invention will have broad therapeutic utility in facilitatingthe treatment of a wide range of inherited and acquired diseases andmedical conditions including, without limitation, hematologic diseases,cardiopulmonary diseases, endocrinological diseases, immunologicaldiseases, neoplasias, and the like.

Without intending to limit it in any manner, the present invention willbe more fully described by the following examples.

MATERIALS AND METHODS

DNA molecules encoding three different complement inhibitor moleculeswere each fused with DNA molecules encoding the MoMLV ecotropic gp70molecule of plasmid pCee (Muenchau et al., 1992) to engineer chimericDNA molecules encoding chimeric proteins. Each fusion was designed sothat the N-terminal region containing the receptor binding domain ofgp70 was removed and replaced with a complement inhibitor sequence.These chimeric DNA molecules were constructed in vectors designed forexpression in mammalian cells. Once generated, the chimeric complementinhibitor/gp70 expression vectors were co-transfected into theGPE86-LXSN3 producer cell line (see below) with plasmid pTH at a 50 to 1ratio. Plasmid pTH (TH stands for TK-hygro) was derived from plasmidp291 (Yates et al., 1985) by digestion of p291 with EcoRV and SspI,purification of the resulting Hph containing fragment, and self ligationof the purified fragment. The resulting plasmid contains sequencesencoding the hygromycin resistance gene Hph, expression of which isdriven by a TK (thymidine kinase) promoter.

Transfectants were selected for approximately 2 weeks in D10 medium (seebelow) containing 150-200 μg/ml of active hygromycin B. Clones wereisolated using cloning rings and vector particles generated fromexpanded clones were assayed for complement resistance.

Construction of the CCPH/gp70E fusion. Plasmid pCDNAI-Amp-Flag-CCPH wasprepared as follows. An mCCPH encoding cDNA fragment was directionallysubcloned into the mammalian expression vector, pcDNAI-AMP (InvitrogenCorporation, San Diego, Calif.) as an EcoRI and NotI restrictionfragment of the mCCPH subclone, pHVS-A11-mCCPH (also referred to aspKS-/mCCPH, ATCC #69178). A 5' FLAG tagged version of mCCPHincorporating a CD59 leader peptide encoding sequence was constructed bythe following steps. A 5' FLAG/mCCPH DNA fragment was prepared utilizingthe EcoRI/NotI restriction fragment of pKS/mCCPH (described above) astemplate in a polymerase chain reaction (PCR). The 5' primer for the PCRwas 5' GCCGGCCTGC AGGACTACAA AGACGATGAC GATAAA TTAA GCTGTCCTACACGTAACCAG-3' (SEQ ID NO:1), where the underlined sequences represent aunique PstI site that was used for cloning purposes and where theitalicized type indicates the FLAG epitope encoding sequence. The 3'primer for the PCR was 5'-CTTCCATTTA AAAGATCTTG CGG-3' (SEQ ID NO: 2),where the underlined sequence represents a unique BglII site used forcloning purposes that corresponds to a BglII site located within CCPH.The 5'FLAG mCCPH DNA fragment generated in this PCR was cloned into thepCRII vector (Invitrogen Corporation, San Diego, Calif.) and sequencedto confirm that the fragment contained the sequence set forth in SEQ IDNO: 3. A CD59 leader-FLAG-mCCPH clone was then constructed by digestingthe mCCPH-pcDNAI-Amp plasmid with BamHI and BglII and purifying theresulting approximately 5870 bp fragment containing the bulk of themCCPH-pcDNAI-Amp plasmid separate from the approximately 250 bp fragmentcontaining native 5' coding sequences of mCCPH. Subsequently, the 5'FLAGmCCPH fragment and the human CD59 leader cDNA fragment (BamHI-PstI) weredirectionally cloned in a three fragment ligation reaction with thepurified approximately 5870 bp BamHI-BglII CCPH fragment. Competent E.coli cells were transformed with the ligation product, miniprep plasmidDNAs were prepared, and a plasmid containing the desired construction(i.e., a 5' FLAG tagged version of mCCPH incorporating a CD59 leaderpeptide encoding sequence) was identified by gel analysis of restrictiondigests and designated pCDNAI-Amp-Flag-CCPH.

Plasmid pCDNAI-Amp-Flag-CCPH was double digested with NdeI and EcoRV,and an approximately 1300 bp fragment isolated and ligated to a fragmentof pCee that was prepared as follows. Plasmid pCee (Muenchau et al.,1992) was digested with AccI and the ends were filled in using theKlenow fragment of DNA polymerase I and nucleotide triphosphates. TheDNA was then digested with NdeI and electrophoresed, and a resultingapproximately 4500 bp fragment, was isolated from the agarose gel.Competent E. coli cells were transformed with the product of theligation of the pCDNAI-Amp-Flag-CCPH fragment and the pCee fragment,miniprep plasmid DNAs were prepared, and appropriate plasmids (i.e.,those containing the Flag-CD59 leader-CCPH-gp70 fusion construction)were identified by gel analysis of restriction digests. The CCPH/gp70junction of an appropriate plasmid was sequenced to confirm that itincluded the sequence set forth in SEQ ID NO: 4, demonstrating that anin frame gene fusion of CCPH with gp70 had indeed been created. Thisplasmid, CCPH/gp70E, has been deposited with the ATCC and given thedesignation 69650.

CD59/gp70E Plasmid Construction. Plasmid pCDGPI#1-pCDNAI-Amp (ATCC#69564) (see copending U.S. patent application Ser. No. 08/205,508,entitled "Chimeric Complement Inhibitor Molecules, filed on Mar. 3,1994) was digested with EagI. Ends were partially filled with the Klenowfragment of DNA polymerase I and only dGTP. The DNA was then digestedwith NdeI and an approximately 780 bp CD59 fragment was isolated.Plasmid pCee was digested with BspEI and its ends were filled with theKlenow fragment of DNA polymerase I and only dCTP. The plasmid DNA wasthen digested with NdeI and an approximately 4750 bp pCee NdeI/BspEIpartial fill fragment was isolated. This fragment was then ligated withthe approximately 780 bp CD59 fragment. Competent E. coli. cells weretransformed with the ligation product, miniprep plasmid DNAs wereprepared, and appropriate plasmids (i.e., those containing the desiredconstruction) were identified by gel analysis of restriction digests.The CD59/gp70 junction of an appropriate plasmid was sequenced toconfirm that it included the sequence set forth in SEQ ID NO: 5,demonstrating that an in frame gene fusion had indeed been created. Thisplasmid, CD59/gp70E, has been deposited with the ATCC and given thedesignation 69652.

DAF/gp70E Plasmid Construction. Plasmid pDC#1-pCDNAI-Amp (ATCC #69563)(see copending U.S. patent application Ser. No. 08/205,508, entitled"Chimeric Complement Inhibitor Molecules, filed on Mar. 3, 1994) wasdouble digested with NdeI and BsmI. An approximately 1110 bp fragmentwas isolated. This 1110 bp NdeI/BsmI fragment contains sequencesencoding DAF. Plasmid pCee was digested with PflM1 and treated with aten fold excess of calf intestinal phosphatase (CIP, New EnglandBiolabs, Beverly, Mass.) to allow the contaminating exonuclease activitypresent in commercial preparations of calf intestinal phosphatase toremove the 3' hydroxyl guanine nucleotide. The CIP treated DNA was thendigested with NdeI and the resulting approximately 4600 bp fragment wasisolated and ligated to the approximately 1110 bp NdeI/BsmI fragment.Competent E. coli cells were transformed with the ligation product,miniprep plasmid DNAs were prepared, and appropriate plasmids (i.e.,those containing the DAF gp70 construction) were identified by gelanalysis of restriction digests. The DAF/gp70 junction of an appropriateplasmid was sequenced to confirm that it included the sequence set forthin SEQ ID NO: 6, demonstrating that an in frame gene fusion had indeedbeen created. This plasmid, DAF/gp70E, has been deposited with the ATCCand given the designation 69651.

Transfection and Expression of CIM/gp70E Chimeras. The producer cellline GPE86-LXSN3 was generated by transducing GPE86 packaging cells(Markowitz et al., 1988) with vector particles bearing retroviral vectorLXSN sequences (Miller and Rosman, 1989) and selecting for neomycinresistance and high titer. Colonies were cloned out, and GPE86-LXSNclone #3 having a NeoR titer of approximately 2×10⁶ CFU/ml was chosenfor further use.

GPE86-LXSN3 producer cells were co-transfected with a 50 to 1 ratio bymass of CIM/gp70E and plasmid pTH, described above.

Approximately 30 colonies were cloned out and expanded for eachCIM/gp70E construct. Vector particle supernatants, less than 24 hoursold, were collected and stored at -70° C. until assayed for complementresistance.

Vector Particles, Blood and Sera. Vector particles which had been storedfrozen at -70° C. were thawed at 37° C. and used in the complementresistance titer assay. Human blood was drawn from healthy volunteers inthe presence of 25 units/ml heparin to prevent clotting. Human serum wasobtained from Sigma Chemical Company, St. Louis, Mo. Human serum wasalso obtained from healthy donors and prepared as described in Welsh etal., 1976.

Complement Resistance Titer Assay. Generally, 100 μl of thawed vectorparticle supernatant was mixed with an equal volume of heat inactivated(56° C. for 30 minutes) or active complement containing human serum(undiluted or diluted in PBS supplemented with 0.15 mM CaCl₂ and 0.5 mMMgCl₂). This mixture was then incubated at 37° C. for 30 to 60 minutesto allow complement to act upon the vector particles. 100 μl of thevector particle/serum mixture was then titered by analyzing transductionof NIH/3T3 cells using G418 resistance as a marker as described below.Experiments with whole human blood were performed similarly except that,after the 30 to 60 minute incubation at 37° C., samples were centrifugedat 3000×g for 10 minutes at 4° C. prior to transduction of NIH 3T3cells.

Serial dilutions of RVVP samples were assayed for titer of transducingRVVPs on NIH/3T3 cells (ATCC designation CCL 163). 2.5×10⁴ cells wereplated in 2 ml of DMEM containing 10% FBS (referred to herein as D10) inwells of 6-well plates. The following day, medium in each well wasreplaced with 2 ml of D10 containing 8 mg/ml of polybrene. The RVVPsample was then added. Ten-fold or 100-fold serial dilutions were madefrom the original well and added to adjacent wells. The plates were thenincubated for 24 hours. Medium was then once again replaced with 2 ml ofthe D10, in this case containing 500 mg/ml of G418 (active).

Selection was accomplished by incubation in the G418 containing D10 for7-12 days with 2 changes of medium during this period. Following thisselection, medium was removed and surviving colonies were fixed andstained for 15 minutes with a saturated solution of methylene blue inmethanol followed by a brief rinse with water.

Example 1: RVVPs expressing complement inhibitor activities areprotected from inactivation by human serum.

LXSN RVVPs were prepared in the parent GPE86-LXSN3 producer cellsdescribed above. RVVPs were also prepared from clones of the CIM/gp70transfected GPE86-LXSN producers cells described above that had beenselected in hygromycin B. RVVPs from unmodified GPE86-LXSN3 producercells, as well as from the cells transfected with the DAF/GP70,CCPH/GP70, and CD59/GP70 chimeric fusion expression vectors, wereassayed in 40% human serum (in buffer) using the complement resistancetiter assay as described above. As shown in FIG. 1, RVVPs produced bycells expressing the chimeric proteins encoded by each chimeric vectorwere substantially protected from complement inactivation as compared toRVVPs produced by unmodified GPE86-LXSN3 producer cells.

Example 2: RVVPs expressing a complement inhibitor activity areprotected from inactivation by human whole blood.

RVVPs prepared from the parent GPE86-LXSN3 producer cells describedabove and from LXSN3/DAF34 producer cells (expressing the chimericDAF/gp70 protein encoded by plasmid DAF/gp70E described above) wereassayed in whole human blood using the complement resistance titer assayas described above. As shown in FIG. 2, RVVPs produced by cloneLXSN3/DAF34 were substantially protected from complement inactivation ascompared to RVVPs produced by unmodified GPE86-LXSN3 producer cells.

DEPOSITS

Plasmids pKS-/mCCPH, CCPH/gp70E, pCDGPI#1-pCDNAI-Amp, CD59/gp70E andDAF/gp70E, discussed above, have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., 20852, UnitedStates of America, in E. coli and have been assigned the designations69178, 69650, 69564, 69652, and 69651 respectively. These deposits weremade under the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for the Purposes of Patent Procedure (1977).

Throughout this application, various publications, patents, and patentapplications have been referred to. The teachings and disclosures ofthese publications, patents, and patent applications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which the present invention pertains.

Although preferred and other embodiments of the invention have beendescribed herein, other embodiments may be perceived by those skilled inthe art without departing from the scope of the invention as defined bythe following claims.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 bases                                                          (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: 5'Flag-mCCPH 5'PCR primer                                    (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCCGGCCTGCAGGACTACAAAGACGATGACGATAAATTAAGCTGTCCTAC50                          ACGTAACCAG60                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: 5'Flag-mCCPH 3'PCR primer                                    (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: Yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTTCCATTTAAAAGATCTTGCGG23                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 239 base pairs                                                    (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: CCPH PCR product                                             (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTGCAGGACTACAAAGACGATGACGATAAATTAAGCTGTCCTACACGTAA50                          CCAGTATGTTTCTGTCAAATATGTGAATCTAACTAACTATTCAGGCCCGT100                         ATCCAAACGGGACAACGCTACACGTGACATGCCGTGAAGGATATGCAAAA150                         AGACCAGTACAAACTGTTACATGCGTCAATGGTAACTGGACTGTACCTAA200                         AAAGTGTCAGAAAAAGAAATGTTCTACACCGCAAGATCT239                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Circular                                                        (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: CCPH/gp70E Junction                                          (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TGTATGAAGATAGACGGAGCC21                                                       CysMetLysIleAspGlyAla                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Circular                                                        (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: CD59/gp70E Junction                                          (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GGTGGAGCGGCCGGACAAGAT21                                                       GlyGlyAlaAlaGlyGlnAsp                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Circular                                                        (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: DAF/gp70E Junction                                           (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCTGAATGCAGTGGGACTCCT21                                                       ProGluCysSerGlyThrPro                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1407 bases                                                        (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA to genomic RNA                                       (A) DESCRIPTION: Ecotropic gp70 protein                                       (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Shinnick, T. M.                                                  Lerner, R. A.                                                                 Sutcliffe, J. G.                                                              (B) TITLE: Nucleotide sequence of                                             Moloney murine leukemia virus.                                                (C) JOURNAL: Nature                                                           (D) VOLUME: 293                                                               (F) PAGES: 543-548                                                            (G) DATE: 1981                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATGGCGCGTTCAACGCTCTCAAAACCCCTTAAAAATAAGGTT42                                  MetAlaArgSerThrLeuSerLysProLeuLysAsnLysVal                                    1510                                                                          AACCCGCGAGGCCCCCTAATCCCCTTAATTCTTCTGATGCTC84                                  AsnProArgGlyProLeuIleProLeuIleLeuLeuMetLeu                                    152025                                                                        AGAGGGGTCAGTACTGCTTCGCCCGGCTCCAGTCCTCATCAA126                                 ArgGlyValSerThrAlaSerProGlySerSerProHisGln                                    303540                                                                        GTCTATAATATCACCTGGGAGGTAACCAATGGAGATCGGGAG168                                 ValTyrAsnIleThrTrpGluValThrAsnGlyAspArgGlu                                    455055                                                                        ACGGTATGGGCAACTTCTGGCAACCACCCTCTGTGGACCTGG210                                 ThrValTrpAlaThrSerGlyAsnHisProLeuTrpThrTrp                                    606570                                                                        TGGCCTGACCTTACCCCAGATTTATGTATGTTAGCCCACCAT252                                 TrpProAspLeuThrProAspLeuCysMetLeuAlaHisHis                                    7580                                                                          GGACCATCTTATTGGGGGCTAGAATATCAATCCCCTTTTTCT294                                 GlyProSerTyrTrpGlyLeuGluTyrGlnSerProPheSer                                    859095                                                                        TCTCCCCCGGGGCCCCCTTGTTGCTCAGGGGGCAGCAGCCCA336                                 SerProProGlyProProCysCysSerGlyGlySerSerPro                                    100105110                                                                     GGCTGTTCCAGAGACTGCGAAGAACCTTTAACCTCCCTCACC378                                 GlyCysSerArgAspCysGluGluProLeuThrSerLeuThr                                    115120125                                                                     CCTCGGTGCAACACTGCCTGGAACAGACTCAAGCTAGACCAG420                                 ProArgCysAsnThrAlaTrpAsnArgLeuLysLeuAspGln                                    130135140                                                                     ACAACTCATAAATCAAATGAGGGATTTTATGTTTGCCCCGGG462                                 ThrThrHisLysSerAsnGluGlyPheTyrValCysProGly                                    145150                                                                        CCCCACCGCCCCCGAGAATCCAAGTCATGTGGGGGTCCAGAC504                                 ProHisArgProArgGluSerLysSerCysGlyGlyProAsp                                    155160165                                                                     TCCTTCTACTGTGCCTATTGGGGCTGTGAGACAACCGGTAGA546                                 SerPheTyrCysAlaTyrTrpGlyCysGluThrThrGlyArg                                    170175180                                                                     GCTTACTGGAAGCCCTCCTCATCATGGGATTTCATCACAGTA588                                 AlaTyrTrpLysProSerSerSerTrpAspPheIleThrVal                                    185190195                                                                     AACAACAATCTCACCTCTGACCAGGCTGTCCAGGTATGCAAA630                                 AsnAsnAsnLeuThrSerAspGlnAlaValGlnValCysLys                                    200205210                                                                     GATAATAAGTGGTGCAACCCCTTAGTTATTCGGTTTACAGAC672                                 AspAsnLysTrpCysAsnProLeuValIleArgPheThrAsp                                    215220                                                                        GCCGGGAGACGGGTTACTTCCTGGACCACAGGACATTACTGG714                                 AlaGlyArgArgValThrSerTrpThrThrGlyHisTyrTrp                                    225230235                                                                     GGCTTACGTTTGTATGTCTCCGGACAAGATCCAGGGCTTACA756                                 GlyLeuArgLeuTyrValSerGlyGlnAspProGlyLeuThr                                    240245250                                                                     TTTGGGATCCGACTCAGATACCAAAATCTAGGACCCCGCGTC798                                 PheGlyIleArgLeuArgTyrGlnAsnLeuGlyProArgVal                                    255260265                                                                     CCAATAGGGCCAAACCCCGTTCTGGCAGACCAACAGCCACTC840                                 ProIleGlyProAsnProValLeuAlaAspGlnGlnProLeu                                    270275280                                                                     TCCAAGCCCAAACCTGTTAAGTCGCCTTCAGTCACCAAACCA882                                 SerLysProLysProValLysSerProSerValThrLysPro                                    285290                                                                        CCCAGTGGGACTCCTCTCTCCCCTACCCAACTTCCACCGGCG924                                 ProSerGlyThrProLeuSerProThrGlnLeuProProAla                                    295300305                                                                     GGAACGGAAAATAGGCTGCTAAACTTAGTAGACGGAGCCTAC966                                 GlyThrGluAsnArgLeuLeuAsnLeuValAspGlyAlaTyr                                    310315320                                                                     CAAGCCCTCAACCTCACCAGTCCTGACAAAACCCAAGAGTGC1008                                GlnAlaLeuAsnLeuThrSerProAspLysThrGlnGluCys                                    325330335                                                                     TGGTTGTGTCTAGTAGCGGGACCCCCCTACTACGAAGGGGTT1050                                TrpLeuCysLeuValAlaGlyProProTyrTyrGluGlyVal                                    340345350                                                                     GCCGTCCTGGGTACCTACTCCAACCATACCTCTGCTCCAGCC1092                                AlaValLeuGlyThrTyrSerAsnHisThrSerAlaProAla                                    355360                                                                        AACTGCTCCGTGGCCTCCCAACACAAGTTGACCCTGTCCGAA1134                                AsnCysSerValAlaSerGlnHisLysLeuThrLeuSerGlu                                    365370375                                                                     GTGACCGGACAGGGACTCTGCATAGGAGCAGTTCCCAAAACA1176                                ValThrGlyGlnGlyLeuCysIleGlyAlaValProLysThr                                    380385390                                                                     CATCAGGCCCTATGTAATACCACCCAGACAAGCAGTCGAGGG1218                                HisGlnAlaLeuCysAsnThrThrGlnThrSerSerArgGly                                    395400405                                                                     TCCTATTATCTAGTTGCCCCTACAGGTACCATGTGGGCTTGT1260                                SerTyrTyrLeuValAlaProThrGlyThrMetTrpAlaCys                                    410415420                                                                     AGTACCGGGCTTACTCCATGCATCTCCACCACCATACTGAAC1302                                SerThrGlyLeuThrProCysIleSerThrThrIleLeuAsn                                    425430                                                                        CTTACCACTGATTATTGTGTTCTTGTCGAACTCTGGCCAAGA1344                                LeuThrThrAspTyrCysValLeuValGluLeuTrpProArg                                    435440445                                                                     GTCACCTATCATTCCCCCAGCTATGTTTACGGCCTGTTTGAG1386                                ValThrTyrHisSerProSerTyrValTyrGlyLeuPheGlu                                    450455460                                                                     AGATCCAACCGACACAAAAGA1407                                                     ArgSerAsnArgHisLysArg                                                         465                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1329 bases                                                        (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA to genomic RNA                                       (A) DESCRIPTION: Xenotropic gp70 protein                                      (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Massey, A. C.                                                    Coppola, M. A.                                                                Thomas, C. Y.                                                                 (B) TITLE: Origin of pathogenic                                               determinants of recombinant                                                   murine leukemia viruses:                                                      Analysis of Bxv-1-related                                                     xenotropic viruses from CWD                                                   mice.                                                                         (C) JOURNAL: Journal of Virology                                              (D) VOLUME: 64                                                                (F) PAGES: 5491-5499                                                          (G) DATE: 1990                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATGGAAGGTCCAGCGTTCTCAAAACCCCTTAAAGATAAGATT42                                  MetGluGlyProAlaPheSerLysProLeuLysAspLysIle                                    1510                                                                          AACCCGTGGGGCCCCCTAATAGTTATAGGGATCTTGGTGAGG84                                  AsnProTrpGlyProLeuIleValIleGlyIleLeuValArg                                    152025                                                                        GCAGGAGCCTCGGTACAACGTGACAGCCCTCACCAGGTCTTC126                                 AlaGlyAlaSerValGlnArgAspSerProHisGlnValPhe                                    303540                                                                        AATGTCACTTGGAGAGTTACCAACCTAATGACAGGACAAACA168                                 AsnValThrTrpArgValThrAsnLeuMetThrGlyGlnThr                                    455055                                                                        GCTAACGCTACCTCCCTCCTGGGGACGATGACAGACACCTTC210                                 AlaAsnAlaThrSerLeuLeuGlyThrMetThrAspThrPhe                                    606570                                                                        CCTAAACTATATTTTGACTTGTGTGATTTAGTTGGAGACCAT252                                 ProLysLeuTyrPheAspLeuCysAspLeuValGlyAspHis                                    7580                                                                          TGGGATGACCCAGAACCCGATATTGGAGATGGTTGCCGCTCT294                                 TrpAspAspProGluProAspIleGlyAspGlyCysArgSer                                    859095                                                                        CCCGGGGGAAGAAAAAGATCAAGACTGTATGACTTCTATGTT336                                 ProGlyGlyArgLysArgSerArgLeuTyrAspPheTyrVal                                    100105110                                                                     TGCCCCGGTCATACTGTACCAATAGGGTGTGGAGGGCCGGGA378                                 CysProGlyHisThrValProIleGlyCysGlyGlyProGly                                    115120125                                                                     GAGGGCTACTGTGGCAAATGGGGATGTGAGACCACTGGACAG420                                 GluGlyTyrCysGlyLysTrpGlyCysGluThrThrGlyGln                                    130135140                                                                     GCATACTGGAAGCCATCATCATCATGGGACCTAATTTCCCTT462                                 AlaTyrTrpLysProSerSerSerTrpAspLeuIleSerLeu                                    145150                                                                        AAGCGAGGAAACACTCCTAAGGATCAGGGCCCCTGTTATGAT504                                 LysArgGlyAsnThrProLysAspGlnGlyProCysTyrAsp                                    155160165                                                                     TCCTCGGTCTCCAGTGGCGTCCAGGGTGCCACACCGGGGGGT546                                 SerSerValSerSerGlyValGlnGlyAlaThrProGlyGly                                    170175180                                                                     CGATGCAACCCCCTAGTCTTAGAATTCACTGACGCGGGTAAA588                                 ArgCysAsnProLeuValLeuGluPheThrAspAlaGlyLys                                    185190195                                                                     AAGGCCAGCTGGGATGCCCCCAAAGTTTGGGGACTAAGACTC630                                 LysAlaSerTrpAspAlaProLysValTrpGlyLeuArgLeu                                    200205210                                                                     TACCGATCCACAGGGGCCGACCCGGTGACCCGGTTCTCTTTG672                                 TyrArgSerThrGlyAlaAspProValThrArgPheSerLeu                                    215220                                                                        ACCCGCCAGGTCCTCAATGTAGGACCCCGCGTCCCCATTGGG714                                 ThrArgGlnValLeuAsnValGlyProArgValProIleGly                                    225230235                                                                     CCTAATCCCGTGATCACAGAACAGCTACCCCCCTCCCAACCC756                                 ProAsnProValIleThrGluGlnLeuProProSerGlnPro                                    240245250                                                                     GTGCAGATCATGCTCCCCAGGCCTCCTCATCCTCCTCCTTCA798                                 ValGlnIleMetLeuProArgProProHisProProProSer                                    255260265                                                                     GGCGCGGCCTCTATGGTCCCTGGGGCTCCCCCGCCTTCTCAA840                                 GlyAlaAlaSerMetValProGlyAlaProProProSerGln                                    270275280                                                                     CAACCTGGGACGGGGGACAGGCTGCTAAACCTAGTAAAAGGA882                                 GlnProGlyThrGlyAspArgLeuLeuAsnLeuValLysGly                                    285290                                                                        GCCTATCAAGCACTCAACCTCACCAGTCCTGACAGAACCCAA924                                 AlaTyrGlnAlaLeuAsnLeuThrSerProAspArgThrGln                                    295300305                                                                     GAGTGCTGGTTGTGTCTGGTATCGGGACCCCCCTACTACGAA966                                 GluCysTrpLeuCysLeuValSerGlyProProTyrTyrGlu                                    310315320                                                                     GGGGTTGCCGTCCTAGGTACCTATTCCAACCATACCTCTGCC1008                                GlyValAlaValLeuGlyThrTyrSerAsnHisThrSerAla                                    325330335                                                                     CCAGCTAACTGCTCCGTGGCCTCCCAACACAAGCTGACCCTG1050                                ProAlaAsnCysSerValAlaSerGlnHisLysLeuThrLeu                                    340345350                                                                     TCCGAAGTGACCGGGCAGGGACTCTGCGTAGGAGCAGTTCCC1092                                SerGluValThrGlyGlnGlyLeuCysValGlyAlaValPro                                    355360                                                                        AAAACCCATCAGGCCCTGTGTAATACCACCCAGAAGGCGAGC1134                                LysThrHisGlnAlaLeuCysAsnThrThrGlnLysAlaSer                                    365370375                                                                     GACGGGTCCTACTATCTGGCTGCTCCCGCCGGGACCATCTGG1176                                AspGlySerTyrTyrLeuAlaAlaProAlaGlyThrIleTrp                                    380385390                                                                     GCTTGCAACACCGGGCTCACTCCCTGCCTATCTACCACTGTA1218                                AlaCysAsnThrGlyLeuThrProCysLeuSerThrThrVal                                    395400405                                                                     CTCAACCTCACCACCGATTACTGTGTCCTGGTTGAGCTCTGG1260                                LeuAsnLeuThrThrAspTyrCysValLeuValGluLeuTrp                                    410415420                                                                     CCAAAGGTGACCTACCACTCCCCTGGTTATGTTTATGACCAG1302                                ProLysValThrTyrHisSerProGlyTyrValTyrAspGln                                    425430                                                                        TTTGAGAGAAAAACCAAATATAAAAGA1329                                               PheGluArgLysThrLysTyrLysArg                                                   435440                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1374 bases                                                        (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA to genomic RNA                                       (A) DESCRIPTION: Amphotropic gp70 protein                                     (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Ott, D.                                                          Friedrich, R.                                                                 Rein, A.                                                                      (B) TITLE: Sequence analysis of amphotropic                                   and 10A1 murine leukemia                                                      viruses: Close relationship to                                                mink cell focus-inducing                                                      viruses.                                                                      (C) JOURNAL: Journal of Virology                                              (D) VOLUME: 64                                                                (F) PAGES: 757-766                                                            (G) DATE: 1990                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATGGCG6                                                                       MetAla                                                                        CGTTCAACGCTCTCAAAACCCCCTCAAGATAAGATTAACCCG48                                  ArgSerThrLeuSerLysProProGlnAspLysIleAsnPro                                    51015                                                                         TGGAAGCCCTTAATAGTCATGGGAGTCCTGTTAGGAGTAGGG90                                  TrpLysProLeuIleValMetGlyValLeuLeuGlyValGly                                    202530                                                                        ATGGCAGAGAGCCCCCATCAGGTCTTTAATGTAACCTGGAGA132                                 MetAlaGluSerProHisGlnValPheAsnValThrTrpArg                                    3540                                                                          GTCACCAACCTGATGACTGGGCGTACCGCCAATGCCACCTCC174                                 ValThrAsnLeuMetThrGlyArgThrAlaAsnAlaThrSer                                    455055                                                                        CTCCTGGGAACTGTACAAGATGCCTTCCCAAAATTATATTTT216                                 LeuLeuGlyThrValGlnAspAlaPheProLysLeuTyrPhe                                    606570                                                                        GATCTATGTGATCTGGTCGGAGAGGAGTGGGACCCTTCAGAC258                                 AspLeuCysAspLeuValGlyGluGluTrpAspProSerAsp                                    758085                                                                        CAGGAACCGTATGTCGGGTATGGCTGCAAGTACCCCGCAGGG300                                 GlnGluProTyrValGlyTyrGlyCysLysTyrProAlaGly                                    9095100                                                                       AGACAGCGGACCCGGACTTTTGACTTTTACGTGTGCCCTGGG342                                 ArgGlnArgThrArgThrPheAspPheTyrValCysProGly                                    105110                                                                        CATACCGTAAAGTCGGGGTGTGGGGGACCAGGAGAGGGCTAC384                                 HisThrValLysSerGlyCysGlyGlyProGlyGluGlyTyr                                    115120125                                                                     TGTGGTAAATGGGGGTGTGAAACCACCGGACAGGCTTACTGG426                                 CysGlyLysTrpGlyCysGluThrThrGlyGlnAlaTyrTrp                                    130135140                                                                     AAGCCCACATCATCGTGGGACCTAATCTCCCTTAAGCGCGGT468                                 LysProThrSerSerTrpAspLeuIleSerLeuLysArgGly                                    145150155                                                                     AACACCCCCTGGGACACGGGATGCTCTAAAGTTGCCTGTGGC510                                 AsnThrProTrpAspThrGlyCysSerLysValAlaCysGly                                    160165170                                                                     CCCTGCTACGACCTCTCCAAAGTATCCAATTCCTTCCAAGGG552                                 ProCysTyrAspLeuSerLysValSerAsnSerPheGlnGly                                    175180                                                                        GCTACTCGAGGGGGCAGATGCAACCCTCTAGTCCTAGAATTC594                                 AlaThrArgGlyGlyArgCysAsnProLeuValLeuGluPhe                                    185190195                                                                     ACTGATGCAGGAAAAAAGGCTAACTGGGACGGGCCCAAATCG636                                 ThrAspAlaGlyLysLysAlaAsnTrpAspGlyProLysSer                                    200205210                                                                     TGGGGACTGAGACTGTACCGGACAGGAACAGATCCTATTACC678                                 TrpGlyLeuArgLeuTyrArgThrGlyThrAspProIleThr                                    215220225                                                                     ATGTTCTCCCTGACCCGGCAGGTCCTTAATGTGGGACCCCGA720                                 MetPheSerLeuThrArgGlnValLeuAsnValGlyProArg                                    230235240                                                                     GTCCCCATAGGGCCCAACCCAGTATTACCCGACCAAAGACTC762                                 ValProIleGlyProAsnProValLeuProAspGlnArgLeu                                    245250                                                                        CCTTCCTCACCAATAGAGATTGTACCGGCTCCACAGCCACCT804                                 ProSerSerProIleGluIleValProAlaProGlnProPro                                    255260265                                                                     AGCCCCCTCAATACCAGTTACCCCCCTTCCACTACCAGTACA846                                 SerProLeuAsnThrSerTyrProProSerThrThrSerThr                                    270275280                                                                     CCCTCAACCTCCCCTACAAGTCCAAGTGTCCCACAGCCACCC888                                 ProSerThrSerProThrSerProSerValProGlnProPro                                    285290295                                                                     CCAGGAACTGGAGATAGACTACTAGCTCTAGTCAAAGGAGCC930                                 ProGlyThrGlyAspArgLeuLeuAlaLeuValLysGlyAla                                    300305310                                                                     TATCAGGCGCTTAACCTCACCAATCCCGACAAGACCCAAGAA972                                 TyrGlnAlaLeuAsnLeuThrAsnProAspLysThrGlnGlu                                    315320                                                                        TGTTGGCTGTGCTTAGTGTCGGGACCTCCTTATTACGAAGGA1014                                CysTrpLeuCysLeuValSerGlyProProTyrTyrGluGly                                    325330335                                                                     GTAGCGGTCGTGGGCACTTATACCAATCATTCCACCGCTCCG1056                                ValAlaValValGlyThrTyrThrAsnHisSerThrAlaPro                                    340345350                                                                     GCCAACTGTACGGCCACTTCCCAACATAAGCTTACCCTATCT1098                                AlaAsnCysThrAlaThrSerGlnHisLysLeuThrLeuSer                                    355360365                                                                     GAAGTGACAGGACAGGGCCTATGCATGGGGGCAGTACCTAAA1140                                GluValThrGlyGlnGlyLeuCysMetGlyAlaValProLys                                    370375380                                                                     ACTCACCAGGCCTTATGTAACACCACCCAAAGCGCCGGCTCA1182                                ThrHisGlnAlaLeuCysAsnThrThrGlnSerAlaGlySer                                    385390                                                                        GGATCCTACTACCTTGCAGCACCCGCCGGAACAATGTGGGCT1224                                GlySerTyrTyrLeuAlaAlaProAlaGlyThrMetTrpAla                                    395400405                                                                     TGCAGCACTGGATTGACTCCCTGCTTGTCCACCACGGTGCTC1266                                CysSerThrGlyLeuThrProCysLeuSerThrThrValLeu                                    410415420                                                                     AATCTAACCACAGATTATTGTGTATTAGTTGAACTCTGGCCC1308                                AsnLeuThrThrAspTyrCysValLeuValGluLeuTrpPro                                    425430435                                                                     AGAGTAATTTACCACTCCCCCGATTATATGTATGGTCAGCTT1350                                ArgValIleTyrHisSerProAspTyrMetTyrGlyGlnLeu                                    440445450                                                                     GAACAGCGTACCAAATATAAAAGA1374                                                  GluGlnArgThrLysTyrLysArg                                                      455                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2096                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Lublin, Douglas M.                                               Atkinson, John P.                                                             (B) TITLE: Decay-Accelerating Factor:                                         Biochemistry, Molecular Biology, and                                          Function                                                                      (C) JOURNAL: Annual Review of Immunology                                      (D) VOLUME: 7                                                                 (F) PAGES: 35-58                                                              (G) DATE: 1989                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCTGCGACTCGGCGGAGTCCCGGCGGCGCGTCCTTGTTCT40                                    AACCCGGCGCGCCATGACCGTCGCGCGGCCGAGCGTGCCC80                                    MetThrValAlaArgProSerValPro                                                   30                                                                            GCGGCGCTGCCCCTCCTCGGGGAGCTGCCCCGGCTGCTGCTG122                                 AlaAlaLeuProLeuLeuGlyGluLeuProArgLeuLeuLeu                                    25-20-15                                                                      CTGGTGCTGTTGTGCCTGCCGGCCGTGTGGGGTGACTGTGGC164                                 LeuValLeuLeuCysLeuProAlaValTrpGlyAspCysGly                                    10- 51                                                                        CTTCCCCCAGATGTACCTAATGCCCAGCCAGCTTTGGAAGGC206                                 LeuProProAspValProAsnAlaGlnProAlaLeuGluGly                                    51015                                                                         CGTACAAGTTTTCCCGAGGATACTGTAATAACGTACAAATGT248                                 ArgThrSerPheProGluAspThrValIleThrTyrLysCys                                    202530                                                                        GAAGAAAGCTTTGTGAAAATTCCTGGCGAGAAGGACTCAGTG290                                 GluGluSerPheValLysIleProGlyGluLysAspSerVal                                    354045                                                                        ACCTGCCTTAAGGGCATGCAATGGTCAGATATTGAAGAGTTC332                                 ThrCysLeuLysGlyMetGlnTrpSerAspIleGluGluPhe                                    5055                                                                          TGCAATCGTAGCTGCGAGGTGCCAACAAGGCTAAATTCTGCA374                                 CysAsnArgSerCysGluValProThrArgLeuAsnSerAla                                    606570                                                                        TCCCTCAAACAGCCTTATATCACTCAGAATTATTTTCCAGTC416                                 SerLeuLysGlnProTyrIleThrGlnAsnTyrPheProVal                                    758085                                                                        GGTACTGTTGTGGAATATGAGTGCCGTCCAGGTTACAGAAGA458                                 GlyThrValValGluTyrGluCysArgProGlyTyrArgArg                                    9095100                                                                       GAACCTTCTCTATCACCAAAACTAACTTGCCTTCAGAATTTA500                                 GluProSerLeuSerProLysLeuThrCysLeuGlnAsnLeu                                    105110115                                                                     AAATGGTCCACAGCAGTCGAATTTTGTAAAAAGAAATCATGC542                                 LysTrpSerThrAlaValGluPheCysLysLysLysSerCys                                    120125                                                                        CCTAATCCGGGAGAAATACGAAATGGTCAGATTGATGTACCA584                                 ProAsnProGlyGluIleArgAsnGlyGlnIleAspValPro                                    130135140                                                                     GGTGGCATATTATTTGGTGCAACCATCTCCTTCTCATGTAAC626                                 GlyGlyIleLeuPheGlyAlaThrIleSerPheSerCysAsn                                    145150155                                                                     ACAGGGTACAAATTATTTGGCTCGACTTCTAGTTTTTGTCTT668                                 ThrGlyTyrLysLeuPheGlySerThrSerSerPheCysLeu                                    160165170                                                                     ATTTCAGGCAGCTCTGTCCAGTGGAGTGACCCGTTGCCAGAG710                                 IleSerGlySerSerValGlnTrpSerAspProLeuProGlu                                    175180185                                                                     TGCAGAGAAATTTATTGTCCAGCACCACCACAAATTGACAAT752                                 CysArgGluIleTyrCysProAlaProProGlnIleAspAsn                                    190195                                                                        GGAATAATTCAAGGGGAACGTGACCATTATGGATATAGACAG794                                 GlyIleIleGlnGlyGluArgAspHisTyrGlyTyrArgGln                                    200205210                                                                     TCTGTAACGTATGCATGTAATAAAGGATTCACCATGATTGGA836                                 SerValThrTyrAlaCysAsnLysGlyPheThrMetIleGly                                    215220225                                                                     GAGCACTCTATTTATTGTACTGTGAATAATGATGAAGGAGAG878                                 GluHisSerIleTyrCysThrValAsnAsnAspGluGlyGlu                                    230235240                                                                     TGGAGTGGCCCACCACCTGAATGCAGAGGAAAATCTCTAACT920                                 TrpSerGlyProProProGluCysArgGlyLysSerLeuThr                                    245250255                                                                     TCCAAGGTCCCACCAACAGTTCAGAAACCTACCACAGTAAAT962                                 SerLysValProProThrValGlnLysProThrThrValAsn                                    260265                                                                        GTTCCAACTACAGAAGTCTCACCAACTTCTCAGAAAACCACC1004                                ValProThrThrGluValSerProThrSerGlnLysThrThr                                    270275280                                                                     ACAAAAACCACCACACCAAATGCTCAAGCAACACGGAGTACA1046                                ThrLysThrThrThrProAsnAlaGlnAlaThrArgSerThr                                    285290295                                                                     CCTGTTTCCAGGACAACCAAGCATTTTCATGAAACAACCCCA1088                                ProValSerArgThrThrLysHisPheHisGluThrThrPro                                    300305310                                                                     AATAAAGGAAGTGGAACCACTTCAGGTACTACCCGTCTTCTA1130                                AsnLysGlySerGlyThrThrSerGlyThrThrArgLeuLeu                                    315320325                                                                     TCTGGGCACACGTGTTTCACGTTGACAGGTTTGCTTGGGACG1172                                SerGlyHisThrCysPheThrLeuThrGlyLeuLeuGlyThr                                    330335                                                                        CTAGTAACCATGGGCTTGCTGACT1196                                                  LeuValThrMetGlyLeuLeuThr                                                      340345                                                                        TAGCCAAAGAAGAGTTAAGAAGAAAATACACACAAGTATACAGACTGTTC1246                        CTAGTTTCTTAGACTTATCTGCATATTGGATAAAATAAATGCAATTGTGC1296                        TCTTCATTTAGGATGCTTTCATTGTCTTTAAGATGTGTTAGGAATGTCAA1346                        CAGAGCAAGGAGAAAAAAGGCAGTCCTGGAATCACATTCTTAGCACACCT1396                        GCGCCTCTTGAAAATAGAACAACTTGCAGAATTGAGAGTGATTCCTTTCC1446                        TAAAAGTGTAAGAAAGCATAGAGATTTGTTCGTATTAAGAATGGGATCAC1496                        GAGGAAAAGAGAAGGAAAGTGATTTTTTTCCACAAGATCTGAAATGATAT1546                        TTCCACTTATAAAGGAAATAAAAAATGAAAAACATTATTTGGATATCAAA1596                        AGCAAATAAAAACCCAATTCAGTCTCTTCTAAGCAAAATTGCTAAAGAGA1646                        GATGACCACATTATAAAGTAATCTTTGGCTAAGGCATTTTCATCTTTCCT1696                        TCGGTTGGCAAAATATTTTAAAGGTAAAACATGCTGGTGAACCAGGGTGT1746                        TGATGGTGATAAGGGAGGAATATAGAATGAAAGACTGAATCTTCCTTTGT1796                        TGCACAAATAGAGTTTGGAAAAAGCCTGTGAAAGGTGTCTTCTTTGACTT1846                        AATGTCTTTAAAAGTATCCAGAGATACTACAATATTAACATAAGAAAAGA1896                        TTATATATTATTTCTGAATCGAGATGTCCATAGTCAAATTTGTAAATCTT1946                        ATTCTTTTGTAATATTTATTTATATTTATTTATGACAGTGAACATTCTGA1996                        TTTTACATGTAAAACAAGAAAAGTTGAAGAAGATATGTGAAGAAAAATGT2046                        ATTTTTCCTAAATAGAAATAAATGATCCCATTTTTTGGTAAAAAAAAAAA2096                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1139 bases                                                        (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (A) DESCRIPTION: CD59 full length cDNA                                        (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Philbrick, W.M.                                                  Palfree, R.G.E                                                                Maher, S.E.                                                                   Bridgett, M.M.                                                                Sirlin S.                                                                     Bothwell, A.L.M.                                                              (B) TITLE: The CD59 antigen is a structural                                   homologue of murine Ly-6 antigens but                                         lacks interferon inducibility.                                                (C) JOURNAL: European Journal of Immunology                                   (D) VOLUME: 20                                                                (F) PAGES: 87-92                                                              (G) DATE: JAN-1990                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CGCAGAAGCGGCTCGAGGCTGGAAGAGGATCCTGGGCGCCGCAGGTTCTG50                          TGGACAATCACAATGGGAATCCAAGGAGGGTCTGTCCTGTTC92                                  MetGlyIleGlnGlyGlySerValLeuPhe                                                25- 20                                                                        GGGCTGCTGCTCGTCCTGGCTGTCTTCTGCCATTCAGGTCAT134                                 GlyLeuLeuLeuValLeuAlaValPheCysHisSerGlyHis                                    15-10-5                                                                       AGCCTGCAGTGCTACAACTGTCCTAACCCAACTGCTGACTGC176                                 SerLeuGlnCysTyrAsnCysProAsnProThrAlaAspCys                                    +1510                                                                         AAAACAGCCGTCAATTGTTCATCTGATTTTGATGCGTGTCTC218                                 LysThrAlaValAsnCysSerSerAspPheAspAlaCysLeu                                    152025                                                                        ATTACCAAAGCTGGGTTACAAGTGTATAACAAGTGTTGGAAG260                                 IleThrLysAlaGlyLeuGlnValTyrAsnLysCysTrpLys                                    303540                                                                        TTTGAGCATTGCAATTTCAACGACGTCACAACCCGCTTGAGG302                                 PheGluHisCysAsnPheAsnAspValThrThrArgLeuArg                                    455055                                                                        GAAAATGAGCTAACGTACTACTGCTGCAAGAAGGACCTGTGT344                                 GluAsnGluLeuThrTyrTyrCysCysLysLysAspLeuCys                                    6065                                                                          AACTTTAACGAACAGCTTGAAAATGGTGGGACATCCTTATCA386                                 AsnPheAsnGluGlnLeuGluAsnGlyGlyThrSerLeuSer                                    707580                                                                        GAGAAAACAGTTCTTCTGCTGGTGACTCCATTTCTGGCAGCA428                                 GluLysThrValLeuLeuLeuValThrProPheLeuAlaAla                                    859095                                                                        GCCTGGAGCCTTCATCCCTAAGTCAACACCAGGAGAGCTTCT470                                 AlaTrpSerLeuHisPro                                                            100                                                                           CCCAAACTCCCCGTTCCTGCGTAGTCCGCTTTCTCTTGCTGCCACATTCT520                         AAAGGCTTGATATTTTCCAAATGGATCCTGTTGGGAAAGAATAAAATTAG570                         CTTGAGCAACCTGGCTAAGATAGAGGGGTCTGGGAGACTTTGAAGACCAG620                         TCCTGCCCGCAGGGAAGCCCCACTTGAAGGAAGAAGTCTAAGAGTGAAGT670                         AGGTGTGACTTGAACTAGATTGCATGCTTCCTCCTTTGCTCTTGGGAAGA720                         CCAGCTTTGCAGTGACAGCTTGAGTGGGTTCTCTGCAGCCCTCAGATTAT770                         TTTTCCTCTGGCTCCTTGGATGTAGTCAGTTAGCATCATTAGTACATCTT820                         TGGAGGGTGGGGCAGGAGTATATGAGCATCCTCTCTCACATGGAACGCTT870                         TCATAAACTTCAGGGATCCCGTGTTGCCATGGAGGCATGCCAAATGTTCC920                         ATATGTGGGTGTCAGTCAGGGACAACAAGATCCTTAATGCAGAGCTAGAG970                         GACTTCTGGCAGGGAAGTGGGGAAGTGTTCCAGATTCCAGATAGCAGGGC1020                        ATGAAAACTTAGAGAGGTACAAGTGGCTGAAAATCGAGTTTTTCCTCTGT1070                        CTTTAAATTTTATATGGGCTTTGTTATCTTCCACTGGAAAAGTGTAATAG1120                        CATACATCAATGGTGTGTT1139                                                       (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1980 base pairs                                                   (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double stranded                                             (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               cDNA to mRNA                                                                  (A) DESCRIPTION: Herpesvirus saimiri mCCPH gene                               (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Herpesvirus saimiri                                             (B) STRAIN: #11                                                               (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT: L-DNA                                                 (B) MAP POSITION: 10546-12525                                                 (C) UNITS: Nucleotide number                                                  (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Albrecht, Jens- Christian                                        Fleckenstein, Bernhard                                                        (B) TITLE: New Member of the Multigene Family of                              Complement Control Proteins in Herpesvirus Saimiri                            (C) JOURNAL: Journal of Virology                                              (D) VOLUME: 66                                                                (E) ISSUE: 6                                                                  (F) PAGES: 3937-3940                                                          (G) DATE: June 1992                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AAGCTTTGTCTTTTAATTCTGTAAGTTTACTTAGGTAATTTAATAACAAA50                          TAAACTTATAAACATATTTTAAGCTTTACTGGTATTGTTGTTTATAACCT100                         TTTTGTTTTATACATAAAAGTTTAAGTAAGATACTTATTTTCTAGTAGCT150                         AGTACGTTGCTTGCTCATTTTTCTAATAGTGTTTATTCTAAAACTTATAT200                         AATTTAAATATAATTTGCAGTAACAGTTTAAAATGTTAAACTTTTGTTAT250                         TTTTAATATGATATATGTTAACAGCTATAGTTGCATTTTATATTTGTGTT300                         TTTATTAATTTAAGAAGGATTAGTAAAATATATTTAACTTTCTGAGAAGA350                         AATTATACAGTTAGCCATGTACACTTTACACTAC384                                         MetTyrThrLeuHisTyr                                                            20-15                                                                         ATTTGTCTTGTTTTGTCATGTGTAATTTATTTTGTATGGACTTTAAGC432                           IleCysLeuValLeuSerCysValIleTyrPheValTrpThrLeuSer                              10-5+1                                                                        TGTCCTACACGTAACCAGTATGTTTCTGTCAAATATGTGAATCTAACT480                           CysProThrArgAsnGlnTyrValSerValLysTyrValAsnLeuThr                              51015                                                                         AACTATTCAGGCCCGTATCCAAACGGGACAACGCTACACGTGACATGC528                           AsnTyrSerGlyProTyrProAsnGlyThrThrLeuHisValThrCys                              202530                                                                        CGTGAAGGATATGCAAAAAGACCAGTACAAACTGTTACATGCGTCAAT576                           ArgGluGlyTyrAlaLysArgProValGlnThrValThrCysValAsn                              35404550                                                                      GGTAACTGGACTGTACCTAAAAAGTGTCAGAAAAAGAAATGTTCTACA624                           GlyAsnTrpThrValProLysLysCysGlnLysLysLysCysSerThr                              556065                                                                        CCGCAAGATCTTTTAAATGGAAGATATACTGTAACTGGTAATTTATAT672                           ProGlnAspLeuLeuAsnGlyArgTyrThrValThrGlyAsnLeuTyr                              707580                                                                        TACGGTTCAGTTATCACTTATACTTGTAATTCAGGCTACAGCTTAATT720                           TyrGlySerValIleThrTyrThrCysAsnSerGlyTyrSerLeuIle                              859095                                                                        GGAAGCACAACATCAGCTTGTTTACTTAAACGAGGTGGTCGTGTTGAC768                           GlySerThrThrSerAlaCysLeuLeuLysArgGlyGlyArgValAsp                              100105110                                                                     TGGACTCCACGACCTCCAATTTGTGACATTAAAAAATGTAAACCTCCT816                           TrpThrProArgProProIleCysAspIleLysLysCysLysProPro                              115120125130                                                                  CCACAAATAGCTAATGGGACTCACACTAATGTCAAAGATTTCTATACT864                           ProGlnIleAlaAsnGlyThrHisThrAsnValLysAspPheTyrThr                              135140145                                                                     TATTTAGATACAGTTACGTACTCATGCAATGACGAAACAAAGTTAACT912                           TyrLeuAspThrValThrTyrSerCysAsnAspGluThrLysLeuThr                              150155160                                                                     TTAACAGGCCCTTCATCGAAACTTTGTTCAGAAACTGGCTCATGGGTA960                           LeuThrGlyProSerSerLysLeuCysSerGluThrGlySerTrpVal                              165170175                                                                     CCTAATGGAGAAACTAAGTGTGAATTTATATTTTGTAAACTACCTCAA1008                          ProAsnGlyGluThrLysCysGluPheIlePheCysLysLeuProGln                              180185190                                                                     GTTGCGAATGCGTACGTTGAAGTTAGAAAGTCAGCTACGAGCATGCAA1056                          ValAlaAsnAlaTyrValGluValArgLysSerAlaThrSerMetGln                              195200205210                                                                  TATTTGCATATAAATGTTAAATGTTATAAAGGATTTATGCTATATGGA1104                          TyrLeuHisIleAsnValLysCysTyrLysGlyPheMetLeuTyrGly                              215220225                                                                     GAAACTCCTAATACGTGTAACCATGGAGTATGGTCTCCAGCTATTCCT1152                          GluThrProAsnThrCysAsnHisGlyValTrpSerProAlaIlePro                              230235240                                                                     GAATGTATGAAGATATCTTCTCCAAAAGGAGACATGCCTGGCATAAAC1200                          GluCysMetLysIleSerSerProLysGlyAspMetProGlyIleAsn                              245250255                                                                     TCAAATGAAGATAATTCTACACCTTCAGGTAGGATATGCAATGGAAAT1248                          SerAsnGluAspAsnSerThrProSerGlyArgIleCysAsnGlyAsn                              260265270                                                                     TGTACAACTAGCATGCCCACTCAAACATATACAATAATTACTGCGCGC1296                          CysThrThrSerMetProThrGlnThrTyrThrIleIleThrAlaArg                              275280285290                                                                  TATACAAGTCACATATATTTCCCTACTGGGAAAACCTATAAACTTCCT1344                          TyrThrSerHisIleTyrPheProThrGlyLysThrTyrLysLeuPro                              295300305                                                                     CGGGGAGTTCTAGTAATTATTCTTACCACAAGCTTTATTATTATTGGA1392                          ArgGlyValLeuValIleIleLeuThrThrSerPheIleIleIleGly                              310315320                                                                     ATAATACTTACTGGAGTGTGTTTACACAGGTGCAGAGTGTGCATGTCC1440                          IleIleLeuThrGlyValCysLeuHisArgCysArgValCysMetSer                              325330335                                                                     GGGCAGTAACTACCCAATTTCTTCATAAATATGAGAATCTCCGTTACAAGTTCTTA1496                  GlyGln                                                                        340                                                                           ACTGGCCATAATCCACACGAGAAGCATCTAAACGAGTATACGCTCCGCAT1546                        CCATCATCATACATATCATCTTCTGGATAGCAAACATCATCATATATAGA1596                        GTCATTTAAACTAGTTGTATTTCTATTACATTCTTCTGAAAGTGGTTGAA1646                        TTTCTTCATAAACTGGGTCATTAGAATAATTGTTTTCTTCTGCTTGTAAT1696                        AGCTTGTGTTTTGCCTTCAAGTGAAATAAAAAAATTTCAGTCATAATTTT1746                        TAAAAAAATATAGAAGTTTCAGTAAATTGTTGTACTTACCAAACAAGCAC1796                        CCATTATTAGTCTTGGTAGCAGCTAGAATAAATCACTTTAAGTTTAAAAG1846                        TTTAAAAATTTCCTGTCAATGTGGTTTGCTTGGAACAAGGTGTCTACTTA1896                        GGATGTGAGTCATTTACTCTTTGAAGTTCAAAAAAAATAACATAGTTAAA1946                        AGCTAAGCCCATTTTCAGTGATATTTAAAAGCTT1980                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1787 base pairs                                                   (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double stranded                                             (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               cDNA to mRNA                                                                  (A) DESCRIPTION: Herpesvirus saimiri sCCPH gene                               (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Herpesvirus saimiri                                             (B) STRAIN: #11                                                               (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT: L-DNA                                                 (B) MAP POSITION: 10546-11773, 11966-12525                                    (C) UNITS: Nucleotide number                                                  (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Albrecht, Jens- Christian                                        Fleckenstein, Bernhard                                                        (B) TITLE: New Member of the Multigene Family of                              Complement Control Proteins in Herpesvirus Saimiri                            (C) JOURNAL: Journal of Virology                                              (D) VOLUME: 66                                                                (E) ISSUE: 6                                                                  (F) PAGES: 3937-3940                                                          (G) DATE: June 1992                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AAGCTTTGTCTTTTAATTCTGTAAGTTTACTTAGGTAATTTAATAACAAA50                          TAAACTTATAAACATATTTTAAGCTTTACTGGTATTGTTGTTTATAACCT100                         TTTTGTTTTATACATAAAAGTTTAAGTAAGATACTTATTTTCTAGTAGCT150                         AGTACGTTGCTTGCTCATTTTTCTAATAGTGTTTATTCTAAAACTTATAT200                         AATTTAAATATAATTTGCAGTAACAGTTTAAAATGTTAAACTTTTGTTAT250                         TTTTAATATGATATATGTTAACAGCTATAGTTGCATTTTATATTTGTGTT300                         TTTATTAATTTAAGAAGGATTAGTAAAATATATTTAACTTTCTGAGAAGA350                         AATTATACAGTTAGCCATGTACACTTTACACTAC384                                         MetTyrThrLeuHisTyr                                                            20-15                                                                         ATTTGTCTTGTTTTGTCATGTGTAATTTATTTTGTATGGACTTTAAGC432                           IleCysLeuValLeuSerCysValIleTyrPheValTrpThrLeuSer                              10-5+1                                                                        TGTCCTACACGTAACCAGTATGTTTCTGTCAAATATGTGAATCTAACT480                           CysProThrArgAsnGlnTyrValSerValLysTyrValAsnLeuThr                              51015                                                                         AACTATTCAGGCCCGTATCCAAACGGGACAACGCTACACGTGACATGC528                           AsnTyrSerGlyProTyrProAsnGlyThrThrLeuHisValThrCys                              202530                                                                        CGTGAAGGATATGCAAAAAGACCAGTACAAACTGTTACATGCGTCAAT576                           ArgGluGlyTyrAlaLysArgProValGlnThrValThrCysValAsn                              35404550                                                                      GGTAACTGGACTGTACCTAAAAAGTGTCAGAAAAAGAAATGTTCTACA624                           GlyAsnTrpThrValProLysLysCysGlnLysLysLysCysSerThr                              556065                                                                        CCGCAAGATCTTTTAAATGGAAGATATACTGTAACTGGTAATTTATAT672                           ProGlnAspLeuLeuAsnGlyArgTyrThrValThrGlyAsnLeuTyr                              707580                                                                        TACGGTTCAGTTATCACTTATACTTGTAATTCAGGCTACAGCTTAATT720                           TyrGlySerValIleThrTyrThrCysAsnSerGlyTyrSerLeuIle                              859095                                                                        GGAAGCACAACATCAGCTTGTTTACTTAAACGAGGTGGTCGTGTTGAC768                           GlySerThrThrSerAlaCysLeuLeuLysArgGlyGlyArgValAsp                              100105110                                                                     TGGACTCCACGACCTCCAATTTGTGACATTAAAAAATGTAAACCTCCT816                           TrpThrProArgProProIleCysAspIleLysLysCysLysProPro                              115120125130                                                                  CCACAAATAGCTAATGGGACTCACACTAATGTCAAAGATTTCTATACT864                           ProGlnIleAlaAsnGlyThrHisThrAsnValLysAspPheTyrThr                              135140145                                                                     TATTTAGATACAGTTACGTACTCATGCAATGACGAAACAAAGTTAACT912                           TyrLeuAspThrValThrTyrSerCysAsnAspGluThrLysLeuThr                              150155160                                                                     TTAACAGGCCCTTCATCGAAACTTTGTTCAGAAACTGGCTCATGGGTA960                           LeuThrGlyProSerSerLysLeuCysSerGluThrGlySerTrpVal                              165170175                                                                     CCTAATGGAGAAACTAAGTGTGAATTTATATTTTGTAAACTACCTCAA1008                          ProAsnGlyGluThrLysCysGluPheIlePheCysLysLeuProGln                              180185190                                                                     GTTGCGAATGCGTACGTTGAAGTTAGAAAGTCAGCTACGAGCATGCAA1056                          ValAlaAsnAlaTyrValGluValArgLysSerAlaThrSerMetGln                              195200205210                                                                  TATTTGCATATAAATGTTAAATGTTATAAAGGATTTATGCTATATGGA1104                          TyrLeuHisIleAsnValLysCysTyrLysGlyPheMetLeuTyrGly                              215220225                                                                     GAAACTCCTAATACGTGTAACCATGGAGTATGGTCTCCAGCTATTCCT1152                          GluThrProAsnThrCysAsnHisGlyValTrpSerProAlaIlePro                              230235240                                                                     GAATGTATGAAGATATCTTCTCCAAAAGGAGACATGCCTGGCATAAAC1200                          GluCysMetLysIleSerSerProLysGlyAspMetProGlyIleAsn                              245250255                                                                     TCAAATGAAGATAATTCTACACCTTCAGGTGCAGAGTGTGCATGTCCG1248                          SerAsnGluAspAsnSerThrProSerGlyAlaGluCysAlaCysPro                              260265270                                                                     GGCAGTAACTACCCAATTTCTTCATAAATATGAGAATCTCCGTTACAAGTTCTT1302                    GlySerAsnTyrProIleSerSer                                                      275280                                                                        AACTGGCCATAATCCACACGAGAAGCATCTAAACGAGTATACGCTCCGCA1352                        TCCATCATCATACATATCATCTTCTGGATAGCAAACATCATCATATATAG1402                        AGTCATTTAAACTAGTTGTATTTCTATTACATTCTTCTGAAAGTGGTTGA1452                        ATTTCTTCATAAACTGGGTCATTAGAATAATTGTTTTCTTCTGCTTGTAA1502                        TAGCTTGTGTTTTGCCTTCAAGTGAAATAAAAAAATTTCAGTCATAATTT1552                        TTAAAAAAATATAGAAGTTTCAGTAAATTGTTGTACTTACCAAACAAGCA1602                        CCCATTATTAGTCTTGGTAGCAGCTAGAATAAATCACTTTAAGTTTAAAA1652                        GTTTAAAAATTTCCTGTCAATGTGGTTTGCTTGGAACAAGGTGTCTACTT1702                        AGGATGTGAGTCATTTACTCTTTGAAGTTCAAAAAAAATAACATAGTTAA1752                        AAGCTAAGCCCATTTTCAGTGATATTTAAAAGCTT1787                                       __________________________________________________________________________

What is claimed is:
 1. A retroviral vector particle expressing acomplement inhibitor activity, wherein the retroviral vector 9article issubstantially protected from inactivation upon exposure to body fluidscontaining complement.
 2. The retroviral vector particle of claim 1wherein the complement inhibitor activity is a DAF activity.
 3. Theretroviral vector particle of claim 1 wherein the complement inhibitoractivity is a CCPH activity.
 4. The retroviral vector particle of claim1 wherein the complement inhibitor activity is a CD59 activity.
 5. Amethod for transducing a cell with a retroviral vector in the presenceof a body fluid containing complement, wherein said method comprisesadministering the retrovital vector particle of claim 1 to the cell. 6.The method of claim 5, wherein the retroviral vector particle isadministered to the cell ex vivo.
 7. A retroviral producer cellproducing the retrovital vector particles of claim
 1. 8. A retroviralvector particle having a chimeric envelope protein, wherein the chimericenvelope protein is an envelope protein with at least a portion of theN-terminal receptor binding domain removed and replaced with a proteindomain having a complement inhibitor activity.
 9. The retroviral vectorparticle of claim 8 wherein the complement inhibitor activity is a DAFactivity.
 10. The retroviral vector particle of claim 8 wherein thecomplement inhibitor activity is a CCPH activity.
 11. The retroviralvector particle of claim 8 wherein the complement inhibitor activity isa CD59 activity.
 12. A retroviral producer cell producing the retroviralvector particles of claim
 8. 13. A method for transducing a cell with aretroviral vector in the presence of a body fluid containing complement,wherein said method comprises administering the retroviral vectorparticle of claim 8 to the cell.
 14. The method of claim 13, wherein theretroviral vector particle is administered to the cell ex vivo.
 15. Amethod for transducing a cell ex vivo with a retroviral vector in thepresence of a body fluid containing complement, wherein said methodcomprises administering the retroviral vector particle of claim 8 to thecell in culture.
 16. A method for transducing a cell ex vivo with aretrovital vector in the presence of a body fluid containing complement,wherein said method comprises administering the retroviral vectorparticle of claim 1 to the cell in culture.
 17. A chimeric retroviralenvelope protein with at least a portion of the N-terminal receptorbinding domain removed and replaced with a protein domain having acomplement inhibitor activity.